Identification and use of target genes for control of plant parasitic nematodes

ABSTRACT

The invention relates to identifying and evaluating target coding sequences for control of plant parasitic nematodes by inhibiting one or more biological functions, and their use. The invention provides methods and compositions for identification of such sequences and for the control of a plant-parasitic nematode population. By feeding one or more recombinant double stranded RNA molecules provided by the invention to the nematode, a reduction in disease may be obtained through suppression of nematode gene expression. The invention is also directed to methods for making transgenic plants that express the double stranded RNA molecules, and the plant cells and plants obtained thereby.

This application is a continuation of U.S. application Ser. No.14/011,647, filed Aug. 27, 2013 (pending), which application is adivisional of U.S. application Ser. No. 11/673,351, filed Feb. 9, 2007,now U.S. Pat. No. 8,519,225, which application claims benefit of andpriority to U.S. Provisional Patent Application 60/772,265, filed Feb.10, 2006, the disclosures of which are herein incorporated by referencein their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to genetic control of plantdisease caused by plant-parasitic nematodes. More specifically, thepresent invention relates to identification of target coding sequences,and to use of recombinant DNA technologies for post-transcriptionallyrepressing or inhibiting expression of target coding sequences in thecells of a plant-parasitic nematode to provide a plant protectiveeffect.

2. Description of Related Art

Plants are subject to multiple potential disease causing agents,including plant-parasitic nematodes, which are active, flexible,elongate, organisms that live on moist surfaces or in liquidenvironments, including films of water within soil and moist tissueswithin other organisms. There are numerous plant-parasitic nematodespecies, including various cyst nematodes (e.g. Heterodera sp.), rootknot nematodes (e.g. Meloidogyne sp.), lesion nematodes (e.g.Pratylenchus sp.), dagger nematodes (e.g. Xiphinema sp.) and stem andbulb nematodes (e.g. Ditylenchus sp.), among others. Tylenchid nematodes(members of the order Tylenchida), including the families Heteroderidae,Meloidogynidae, and Pratylenchidae, are the largest and mosteconomically important group of plant-parasitic nematodes. Otherimportant plant-parasitic nematodes include Dorylaimid nematodes (e.g.Xiphinema sp.), among others. Nematode species grow through a series oflifecycle stages and molts. Typically, there are five stages and fourmolts: egg stage; J1 (i.e. first juvenile stage); M1 (i.e. first molt);J2 (second juvenile stage; sometimes hatch from egg); M2; J3; M3; J4;M4; A (adult). Juvenile (“J”) stages are also sometimes referred to aslarval (“L”) stages. Gene expression may be specific to one or morelifecycle stages.

Some species of nematodes have evolved as very successful parasites ofboth plants and animals and are responsible for significant economiclosses in agriculture and livestock and for morbidity and mortality inhumans. Nematode parasites of plants can inhabit all parts of plants,including roots, developing flower buds, leaves, and stems. Plantparasites are classified on the basis of their feeding habits into thebroad categories migratory ectoparasites, migratory endoparasites, andsedentary endoparasites. Sedentary endoparasites, which include the rootknot nematodes (Meloidogyne) and cyst nematodes (Globodera andHeterodera) induce feeding sites (“syncytia”) and establish long-terminfections within roots that are often very damaging to crops. It isestimated that parasitic nematodes cost the horticulture and agricultureindustries in excess of $78 billion worldwide a year, based on anestimated average 12% annual loss spread across all major crops. Forexample, it is estimated that nematodes cause soybean losses ofapproximately $3.2 billion annually worldwide (Barker et al., 1994).

Compositions, methods, and agents for controlling infestations bynematodes have been provided in several forms. Biological and culturalcontrol methods, including plant quarantines, have been attempted innumerous instances. In some crops, plant resistance genes have beenidentified that allow nematode resistance or tolerance. Chemicalcompositions such as nematocides have typically been applied to soil inwhich plant parasitic nematodes are present. However, there is an urgentneed for safe and effective nematode controls. Factors relating to thedisadvantages of current control strategies include heightened concernfor the sustainability of agriculture, and new government regulationsthat may prevent or severely restrict the use of many availableagricultural chemical antihelminthic agents.

Chemical agents are often not selective, and exert their effects onnon-target organisms, effectively disrupting populations of beneficialmicroorganisms, for a period of time following application of the agent.Chemical agents may persist in the environment and only be slowlymetabolized. Nematocidal soil fumigants such as chloropicrin and methylbromide and related compounds are highly toxic, and methyl bromide hasbeen identified as an ozone-depleting compound. Thus its registrationfor use in the United States is not being renewed. These agents may alsoaccumulate in the water table or the food chain, and in higher trophiclevel species. These agents may also act as mutagens and/or carcinogensto cause irreversible and deleterious genetic modifications. Thus,alternative methods for nematode control, such as genetic methods, areincreasingly being studied.

The organism Caenorhabditis elegans, a bacteriovorous nematode, is themost widely studied nematode genetic model. Public and private databaseshold a wealth of information on its genetics and development, butpractically applying this information for control of plant-parasiticnematodes remains a challenge (McCarter et al. 2003; McCarter 2004). Ithas previously been impractical to routinely identify a large number oftarget genes in nematodes other than C. elegans, such as plant-parasiticnematodes, for subsequent functional analysis e.g. by RNAi analysis.Therefore, there has existed a need for improved methods of identifyingtarget genes, suppression of expression of which leads to control ofnematode infestation.

Many genes in C. elegans have orthologs in metazoan animals includinginsects and vertebrates as well as other nematodes. In recent years, agreatly expanded expressed sequence tag (EST) collection has beengenerated from over 30 parasitic nematode species of plants and animals(Parkinson et al., 2004). As of 2005 there were approximately 560,874nucleotide sequences in Genbank from nematodes other than Caenorhabditisspecies and public projects are underway to generate draft sequences ofMeloidogyne hapla (root knot nematode), Haemonchus contortus (parasiteof sheep), Trichinella spiralis (parasite of humans and other mammals)(430,000 sequence traces submitted), and Pristionchus pacificus (freeliving nematode) (149,000 sequence traces submitted). 20,109 ESTs areavailable from Heterodera glycines representing portions ofapproximately 9,000 genes (see, e.g., U.S. patent application Ser. No.11/360,355, filed Feb. 23, 2006). Conserved genes are expected to oftenretain the same or very similar functions in different nematodes. Thisfunctional equivalence has been demonstrated in some cases bytransforming C. elegans with homologous genes from other nematodes (Kwaet al., 1995; Redmond et al. 2001). Such equivalence has been shown incross phyla comparisons for conserved genes and is expected to be morerobust among species within a phylum.

RNA interference (RNAi) is a process utilizing endogenous cellularpathways whereby a double stranded RNA (dsRNA) specific target generesults in the degradation of the mRNA of interest. In recent years,RNAi has been used to perform gene “knockdown” in a number of speciesand experimental systems, from the nematode C. elegans, to plants, toinsect embryos and cells in tissue culture (Fire et al., 1998; Martinezet al., 2002; McManus and Sharp, 2002). RNAi works through an endogenouspathway including the Dicer protein complex that generates˜21-nucleotide small interfering RNAs (siRNAs) from the original dsRNAand the RNA-induced silencing complex (RISC) that uses siRNA guides torecognize and degrade the corresponding mRNAs. Only transcriptscomplementary to the siRNA are cleaved and degraded, and thus theknock-down of mRNA expression is usually sequence specific. The genesilencing effect of RNAi persists for days and, under experimentalconditions, can lead to a decline in abundance of the targetedtranscript of 90% or more, with consequent decline in levels of thecorresponding protein.

dsRNA-mediated gene suppression by RNAi can be achieved in C. elegans byfeeding, by soaking the nematodes in solutions containing doublestranded or small interfering RNA molecules, and by injection of thedsRNA molecules (Kamath et al., 2001; Maeda et al., 2001. Severallarge-scale surveys of C. elegans genes by RNAi have been performed sothat RNAi knockdown information is available for >90% of C. elegansgenes (Gonczy et al., 2000; Fraser et al., 2000; Piano et al., 2000;Maeda et al., 2001; Kamath et al., 2003; Simmer et al., 2003; Ashrafi etal., 2003; Sonnichsen et al., 2005).

To date, only limited published technical or patent information existson RNAi-mediated gene suppression in plant parasitic nematodes, whereinthe double-stranded (dsRNA) or small interfering (siRNA) molecules aretaken up from artificial growth media (in vitro) or from plant tissue(in planta). RNAi has been observed to function in several parasiticnematodes including the plant parasites Heterodera glycines andGlobodera pallida (Urwin et al., 2002; US Publication US2004/0098761; USPublication US2003/0150017; US Publication US2003/0061626; USPublication US2004/0133943; Fairbairn et al. 2005), Meloidogyne javanica(WO2005/019408), and the mammalian parasites Nippostrongylusbrasiliensis (Hussein et al., 2002), Brugia malayi (Aboobaker et al.,2003), and Onchocerca volvulus (Lustigman et al., 2004). Production ofparasite-specific dsRNA in plant cells has been suggested as a directstrategy for control of plant parasitic nematodes including the soybeancyst nematode, Heterodera glycines (e.g. Fire et al., 1998; USPublication US2004/0098761; WO 03/052110 A2; US PublicationUS2005/0188438). US Publication US2006/0037101 describes use of H.glycines sequences, such as from pas5, to modulate SCN gene expression.However, no systematic method for identifying target nematode genes foruse in such strategies has been reported, and only a limited number ofplant-parasitic nematode genes have been proposed as potential targetsfor RNAi-mediated gene suppression studies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: C. elegans RNAi Phenotype Ranking System

FIG. 2: Results of C. elegans P0 RNAi feeding studies

FIG. 3A-3D: Top 300 List of H. glycines Gene Targets Based on C. elegansorthologs

SUMMARY OF THE INVENTION

The present invention is directed toward compositions and methods forcontrolling diseases caused by plant-parasitic nematodes. The presentinvention provides exemplary nucleic acid compositions that arehomologous to at least a portion of one or more native nucleic acidsequences in a target plant-parasitic nematode. In certain embodiments,the nematode is selected from Heterodera sp., Meloidogyne sp., Globoderasp., Helicotylenchus sp., Ditylenchus sp., Pratylenchus sp.,Paratylenchus sp., Rotylenchus sp., Tylenchulus sp., Tylenchorhynchussp., Hoplolaimus sp., Belonolaimus sp., Anguina sp., Subanguina sp. andNacobbus sp. In particular, the nematode may be a Heterodera sp., suchas H. glycines. Specific examples of such nucleic acids provided by theinvention are given in the attached sequence listing as SEQ IDNOs:301-1026, SEQ ID NOs:1269-1702, and SEQ ID NOs:1920-1929. However,in certain embodiments, the invention does not comprise SEQ ID NOs:525,569, 797, 1293 or 1516.

Thus, in one aspect, the invention provides an isolated polynucleotideselected from the group consisting of: (a) a fragment of at least 21contiguous nucleotides of a nucleic acid sequence of any of SEQ IDNOs:301-1026, SEQ ID NOs:1269-1702, and SEQ ID NOs:1920-1929, as setforth in the sequence listing, wherein uptake by a plant-parasiticnematode of a double stranded ribonucleotide sequence comprising atleast one strand that is complementary to said fragment inhibits thegrowth of said nematode; and (b) a complement of the sequence of (a). Inanother aspect, the invention provides this isolated polynucleotide,further defined as operably linked to a heterologous promoter. However,in certain embodiments, the invention does not comprise SEQ ID NOs:525,569, 797, 1293 or 1516. In yet another aspect, the invention providesthis isolated polynucleotide further defined as comprised on a planttransformation vector. As used herein uptake by a plant-parasiticnematode includes ingestion of one or more sequences by the nematode,for example, by feeding. In specific non-limiting embodiments, uptakemay be achieved by contacting a plant-parasitic nematode with acomposition comprising one or more nucleic acid(s) according to theinvention. For instance, uptake may also be achieved by soaking ofplant-parasitic nematodes with a solution comprising the nucleicacid(s).

The invention is also directed to a double stranded ribonucleotidesequence produced from the expression of the above polynucleotide,wherein the taking up of said ribonucleotide sequence by aplant-parasitic nematode inhibits the growth of said nematode. Theinvention further provides a double stranded ribonucleotide sequenceproduced by preparing a recombinant polynucleotide sequence comprising afirst, a second and a third polynucleotide sequence, wherein the firstpolynucleotide sequence comprises an isolated polynucleotide, uptake ofwhich by a plant-parasitic nematode inhibits the growth, feeding, ordevelopment of said nematode, wherein the third polynucleotide sequenceis linked to the first polynucleotide sequence by the secondpolynucleotide sequence, and wherein the third polynucleotide sequenceis substantially the reverse complement of the first polynucleotidesequence such that the first and the third polynucleotide sequenceshybridize when transcribed into a ribonucleic acid to form the doublestranded ribonucleotide molecule stabilized by the linked secondribonucleotide sequence. Inhibition of nematode growth, feeding, ordevelopment may be accomplished by inhibiting expression of a nucleotidesequence in the plant-parasitic nematode that is substantiallycomplementary to the sequence of the first polynucleotide.

The invention further provides a plant transformation vector comprisingthe above mentioned nucleotide sequence, wherein the sequence isoperably linked to a heterologous promoter functional in a plant cell,and to cells transformed with the vector. The cells may be prokaryoticor eukaryotic. In particular, they may be plant cells. Plants and seedsderived from such transformed plant cells are also contemplated. Theinvention further provides a commodity product produced from such aplant, wherein said commodity product comprises a detectable amount ofthe polynucleotide of claim 1 or a ribonucleotide expressed therefrom.Methods to produce such a commodity product are also contemplated, byobtaining such transformed plants and preparing food or feed from them.In particular, the food or feed is defined as oil, meal, protein,starch, flour or silage.

The invention also relates to methods for controlling a population of aplant-parasitic nematode, such as H. glycines, comprising providing anagent comprising a double stranded ribonucleotide sequence thatfunctions upon being taken up by the nematode to inhibit a biologicalfunction within said nematode, wherein the agent comprises a nucleotidesequence selected from the group consisting of SEQ ID NOs:301-1026, SEQID NOs:1269-1702, and SEQ ID NOs:1920-1929, and complements thereof.However, in certain embodiments, the invention does not relate to use ofSEQ ID NOs:525, 569, 797, 1293 or 1516. The polynucleotide sequence mayexhibit from about 95 to about 100% nucleotide sequence identity alongat least from about 19 to about 25 contiguous nucleotides to a targetcoding sequence derived from said nematode. The target sequence mayencode a protein, the predicted function of which is selected from thegroup consisting of: DNA replication, cell cycle control, transcription,RNA processing, translation, ribosome function, tRNA synthesis, tRNAfunction, protein trafficking, secretion, protein modification, proteinstability, protein degradation, energy production, mitochondrialfunction, intermediary metabolism, cell structure, signal transduction,endocytosis, ion regulation and transport.

The invention further provides a method for reducing the number ofHeterodera feeding sites established in the root tissue of a host plant,comprising providing in the host plant of a Heterodera sp. a transformedplant cell expressing a polynucleotide sequence of any of SEQ IDNOs:301-1026, SEQ ID NOs:1269-1702, and SEQ ID NOs:1920-1929, whereinthe polynucleotide is expressed to produce a double stranded ribonucleicacid that functions upon being taken up by the Heterodera sp. to inhibitthe expression of a target sequence within said nematode and results ina decrease in the number of feeding sites established, relative togrowth on a host lacking the transformed plant cell.

The present invention also relates to a method for improving the yieldof a crop produced from a crop plant subjected to plant-parasiticnematode infection, said method comprising the steps of: a) introducinga polynucleotide selected from SEQ ID NOs:301-1026, SEQ IDNOs:1269-1702, and SEQ ID NOs:1920-1929, into said crop plant; b)cultivating the crop plant to allow the expression of saidpolynucleotide, wherein expression of the polynucleotide inhibitsplant-parasitic nematode infection or growth and loss of yield due toplant-parasitic nematode infection. However, in certain embodiments, theinvention does not comprise use of a polynucleotide selected from thegroup consisting of SEQ ID NOs:525, 569, 797, 1293 or 1516. Inparticular, the crop plant may be soybean (Glycine max), and theplant-parastic nematode is a Tylenchid nematode such as H. glycines.

The invention additionally provides a method for modulating theexpression of a target gene in a plant-parasitic nematode cell, themethod comprising: (a) transforming a plant cell with a vectorcomprising a nucleic acid sequence encoding a dsRNA selected from thegroup consisting of SEQ ID NOs:301-1026, SEQ ID NOs:1269-1702, and SEQID NOs:1920-1929, operatively linked to a promoter and a transcriptiontermination sequence; (b) culturing the transformed plant cell underconditions sufficient to allow for development of a plant cell culturecomprising a plurality of transformed plant cells; (c) selecting fortransformed plant cells that have integrated the nucleic acid sequenceinto their genomes; (d) screening the transformed plant cells forexpression of the dsRNA encoded by the nucleic acid sequence; and (e)selecting a plant cell that expresses the dsRNA. However, in certainembodiments, the invention does not relate to use of SEQ ID NOs:525,569, 797, 1293 or 1516. Plants may also be regenerated from such plantcells. In particular, “modulating expression” may comprise inhibitingexpression.

The invention also contemplates a method of identifying genes likely tobe essential in the lifecycle of a target nematode, comprising: (a)ranking a Caenorhabditis elegans gene according to one or more criteriaselected from the group consisting of: potency of reported RNAiphenotype; level of confidence in the reported phenotype; and likelihoodof effect of RNAi at multiple stages in a nematode's lifecycle, suchthat a phenotype score is obtained, wherein a high rank indicates a C.elegans gene with a higher likelihood of demonstrating a detectable RNAiphenotype compared to the likelihood of such a phenotype in a lowerranked gene; (b) identifying possible orthologs of a C. elegans gene inthe genome of the target nematode by performing a sequence similaritysearch in a protein or nucleic acid database such that a protein ornucleic acid sequence from a nematode other than C. elegans that has athreshold BLAST e-value of e⁻¹⁰ when compared with a C. elegans sequenceis deemed a possible ortholog of the C. elegans sequence; (c)identifying, among the possible orthologs of step (b), those genes froma nematode other than C. elegans that demonstrate a phenotype score instep (a) among the top 3.5% of all C. elegans genes. However, in certainembodiments, the invention does not comprise identification of SEQ IDNOs:525, 569, 797, 1293 or 1516.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of the invention provided to aidthose skilled in the art in practicing the present invention. Those ofordinary skill in the art may make modifications and variations in theembodiments described herein without departing from the spirit or scopeof the present invention.

The present invention provides methods and compositions for geneticcontrol of plant-parasitic nematode infestations. Methods foridentifying genes essential to the lifecycle of a plant-parasiticnematode for use as a target for dsRNA-mediated control of a nematodepopulation are also provided. DNA plasmid vectors encoding dsRNAmolecules are designed to suppress nematode genes essential for growthand development. For example, the present invention provides methods andrecombinant DNA technologies for post-transcriptionally repressing orinhibiting expression of a target coding sequence in a plant-parasiticnematode to provide a protective effect by allowing the plant-parasiticnematode to ingest one or more double stranded or small interferingribonucleic acid (RNA) molecules transcribed from all or a portion of atarget coding sequence, thereby controlling the infection. Therefore,the present invention relates to sequence-specific inhibition ofexpression of coding sequences using double-stranded RNA (dsRNA),including small interfering RNA (siRNA), to achieve the intended levelsof nematode control.

In another aspect, the invention provides a method for evaluating thelikelihood that a gene is useful as a target for dsRNA-mediated genesuppression for the purpose of controlling a nematode population. Onechallenge of achieving parasite control via RNAi is the selection ofappropriate gene targets which will result in the disruption of theparasite's lifecycle following transcript knockdown. Since thethroughput of systems to test candidate genes in Heterodera glycines islimited, prioritization of targets prior to testing is important.RNAi-based functional genomic screens are highly effective filters thatcan narrow target choices by an order of magnitude or more. In certainembodiments the nematode is a plant parasitic nematode. In anotherembodiment, the nematode is a Tylenchid plant-parasitic nematode. Inanother embodiment the nematode is a Heterodera sp. In yet anotherembodiment, the nematode is the soybean cyst nematode (Heteroderaglycines).

A method for inhibiting target gene function within the plant pathogensoybean cyst nematode, Heterodera glycines, is also provided by thepresent invention, and can be accomplished by RNA interference,resulting in disruption of the pathogen's lifecycle. Optimal targetgenes for disruption include life-cycle essential genes where disruptionresults in high penetrance death of the parasite populations or “geneticdeath” by blocking of reproduction with minimal feeding damage to theplant, reduction in number of established feeding sites, and minimalviable escaping worms reaching the next generation. Another aspect ofthe present invention provides the nucleic acids of each of the 300target genes predicted to be essential to H. glycines growth and/ordevelopment (FIG. 3). Features used to predict such targets includeorthology to known C. elegans genes with strong and reproducible RNAinterference phenotypes, and expression pattern in H. glycines.

In yet another aspect of the present invention, a set of isolated andpurified nucleotide sequences as set forth in SEQ ID NOs:301-1026, SEQID NOs:1269-1702, and SEQ ID NOs:1920-1929, is provided. However, incertain embodiments, the invention does not comprise SEQ ID NOs:525,569, 797, 1293 or 1516. The present invention provides a stabilizeddsRNA molecule for the expression of one or more RNAs for inhibition ofexpression of a target gene in a plant-parasitic nematode, expressedfrom these sequences and fragments thereof. A stabilized dsRNA,including a dsRNA or siRNA molecule can comprise at least two codingsequences that are arranged in a sense and an antisense orientationrelative to at least one promoter, wherein the nucleotide sequence thatcomprises a sense strand and an antisense strand are linked or connectedby a spacer sequence of at least from about five to about one thousandnucleotides, wherein the sense strand and the antisense strand may be adifferent length, and wherein each of the two coding sequences shares atleast 80% sequence identity, at least 90%, at least 95%, at least 98%,or 100% sequence identity, to any one or more nucleotide sequence(s) setforth in SEQ ID NOs:301-1026, SEQ ID NOs:1269-1702, and SEQ IDNOs:1920-1929.

In yet another aspect, the invention provides recombinant DNA constructscomprising a nucleic acid molecule encoding a dsRNA molecule describedherein. The dsRNA may be formed by transcription of one strand of thedsRNA molecule from a nucleotide sequence which is at least from about80% to about 100% identical to a nucleotide sequence selected from thegroup consisting of SEQ ID NOs:301-1026, SEQ ID NOs:1269-1702, and SEQID NOs:1920-1929. Such recombinant DNA constructs may be defined asproducing dsRNA molecules capable of inhibiting the expression ofendogenous target gene(s) in a plant-parasitic nematode cell uponingestion. The construct may comprise a nucleotide sequence of theinvention operably linked to a promoter sequence that functions in thehost cell such as a plant cell. Such a promoter may be tissue-specificand may, for example, be specific to a tissue type which is the subjectof plant-parasitic nematode attack. In the case of a root or foliarpathogen, respectively for example, it may be desired to use a promoterproviding root or leaf-preferred expression, respectively.

Nucleic acid constructs in accordance with the invention may comprise atleast one non-naturally occurring nucleotide sequence that can betranscribed into a single stranded RNA capable of forming a dsRNAmolecule in vivo through intermolecular hybridization. Such dsRNAsequences self assemble and can be provided in the nutrition source of aplant-parasitic nematode to achieve the desired inhibition.

A recombinant DNA construct may comprise two different non-naturallyoccurring sequences which, when expressed in vivo as dsRNA sequences andprovided in the tissues of the host plant of a plant-parasitic nematode,inhibit the expression of at least two different target genes in theplant-parasitic nematode. In certain embodiments, at least 2, 3, 4, 5,6, 8 or 10 or more different dsRNAs are produced in a cell, or plantcomprising the cell, that have a nematode-inhibitory effect. The dsRNAsmay be expressed from multiple constructs introduced in differenttransformation events or could be introduced on a single nucleic acidmolecule. The dsRNAs may be expressed using a single promoter ormultiple promoters. In one embodiment of the invention, single dsRNAsare produced that comprise nucleic acids homologous to multiple lociwithin a plant-parasitic nematode.

In still yet another aspect, the invention provides a recombinant hostcell having in its genome at least one recombinant DNA sequence that istranscribed to produce at least one dsRNA molecule that functions wheningested by a plant-parasitic nematode to inhibit the expression of atarget gene in the nematode. The dsRNA molecule may be encoded by any ofthe nucleic acids described herein and as set forth in the sequencelisting. The present invention also provides a transformed plant cellhaving in its genome at least one recombinant DNA sequence describedherein. Transgenic plants comprising such a transformed plant cell arealso provided, including progeny plants of any generation, seeds, andplant products, each comprising the recombinant DNA. The dsRNA moleculesof the present invention may be found in the transgenic plant cell, forinstance in the cytoplasm. They may also be found in an apoplasticspace.

The invention also provides one or more stabilization sequences, or“clamps”, which may be unrelated to the gene of interest. A clamppreferably comprises a GC rich region that serves to thermodynamicallystabilize the dsRNA molecule, and may increase gene silencing.

Further provided by the invention is a fragment of a nucleic acidsequence selected from the group consisting of SEQ ID NOs:301-1026, SEQID NOs:1269-1702, and SEQ ID NOs:1920-1929. The fragment may be definedas causing the death, growth inhibition, or cessation of infection orfeeding by a plant-parasitic nematode, when expressed as a dsRNA andtaken up by the nematode. The fragment may, for example, comprise atleast about 19, 21, 23, 25, 40, 60, 80, 100, 125 or more contiguousnucleotides of any one or more of the sequences in SEQ ID NOs:301-1026,SEQ ID NOs:1269-1702, and SEQ ID NOs:1920-1929, or a complement thereof.However, in certain embodiments, the invention does not comprise afragment or complement of SEQ ID NOs:525, 569, 797, 1293 or 1516. Onebeneficial DNA segment for use in the present invention is at least fromabout 19 to about 23, or about 23 to about 100 nucleotides, but lessthan about 2000 nucleotides, in length. Particularly useful will bedsRNA sequences including about 23 to about 300 nucleotides homologousto a nematode target sequence. The invention also provides a ribonucleicacid expressed from any of such sequences including a dsRNA. A sequenceselected for use in expression of a gene suppression agent can beconstructed from a single sequence derived from one or more targetplant-parasitic nematode species and intended for use in expression ofan RNA that functions in the suppression of a single gene or gene familyin the one or more target pathogens, or that the DNA sequence can beconstructed as a chimera from a plurality of DNA sequences.

In another embodiment, the invention provides a method for modulatingexpression of a target gene in a nematode cell, the method comprising:(a) transforming a plant cell with a vector comprising a nucleic acidsequence encoding a dsRNA operatively linked to a promoter and atranscription termination sequence; (b) culturing the transformed plantcell under conditions sufficient to allow for development of a plantcell culture comprising a plurality of transformed plant cells; (c)selecting for transformed plant cells that have integrated the vectorinto their genomes; (d) screening the transformed plant cells forexpression of the dsRNA encoded by the vector; (e) selecting a plantcell that expresses the dsRNA; (f) optionally regenerating a plant fromthe plant cell that expresses the dsRNA; whereby expression of the genein the plant is sufficient to modulate the expression of a target genein a cell of a plant parasitic nematode that contacts the transformedplant or plant cell. Modulation of gene expression may include partialor complete suppression of such expression.

In yet another aspect, the invention provides a method for suppressionof gene expression in a plant-parasitic nematode, comprising theprovision in the tissue of the host of the nematode a gene-suppressiveamount of at least one dsRNA molecule transcribed from a nucleotidesequence as described herein, at least one segment of which iscomplementary to an mRNA sequence within the cells of theplant-parasitic nematode. The method may further comprise observing thedeath or growth inhibition of the plant-parasitic nematode, and thedegree of host symptomatology. A dsRNA molecule, including its modifiedform such as an siRNA molecule, ingested by a pathogenic microorganismin accordance with the invention may be at least from about 80, 81, 82,83, 84, 85, 86, 87, 88 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, orabout 100% identical to a RNA molecule transcribed from a nucleotidesequence selected from the group consisting of SEQ ID NOs:301-1026, SEQID NOs:1269-1702, and SEQ ID NOs:1920-1929.

Isolated and substantially purified nucleic acid molecules including,but not limited to, non-naturally occurring nucleotide sequences andrecombinant DNA constructs for transcribing dsRNA molecules of thepresent invention are therefore provided, which suppress or inhibit theexpression of an endogenous coding sequence or a target coding sequencein the plant-parasitic nematode when introduced thereto. Transgenicplants that (a) contain nucleotide sequences encoding the isolated andsubstantially purified nucleic acid molecules and the non-naturallyoccurring recombinant DNA constructs for transcribing the dsRNAmolecules for controlling plant-parasitic nematode infections, and (b)display resistance and/or enhanced tolerance to the infections, are alsocontemplated. Compositions containing the dsRNA nucleotide sequences ofthe present invention for use in topical applications onto plants oronto animals or into the environment of an animal to achieve theelimination or reduction of plant-parasitic nematode infection are alsoincluded.

cDNA sequences encoding proteins or parts of proteins essential forsurvival, such as amino acid sequences involved in various metabolic orcatabolic biochemical pathways, cell division, reproduction, energymetabolism, digestion, and the like may be selected for use in preparingdouble stranded RNA molecules to be provided in the host plant of aplant-parasitic nematode. As described herein, ingestion of compositionsby a target organism containing one or more dsRNAs, at least one segmentof which corresponds to at least a substantially identical segment ofRNA produced in the cells of the target pathogen, can result in thedeath or other inhibition of the target. These results indicate that anucleotide sequence, either DNA or RNA, derived from a plant-parasiticnematode can be used to construct plant cells resistant to infestationby the nematode. The host plant of the nematode, for example, can betransformed to contain one or more of the nucleotide sequences derivedfrom the nematode as provided herein. The nucleotide sequencetransformed into the host may encode one or more RNAs that form into adsRNA sequence in the cells or biological fluids within the transformedhost, thus making the dsRNA available if/when the plant-parasiticnematode forms a nutritional relationship with the transgenic host. Thismay result in the suppression of expression of one or more genes in thecells of the plant-parasitic nematode and ultimately death or inhibitionof its growth or development.

The present invention relates generally to genetic control ofplant-parasitic nematodes in host organisms. More particularly, thepresent invention includes methods for delivery of nematode controlagents to plant-parasitic nematodes. Such control agents cause, directlyor indirectly, an impairment in the ability of the plant-parasiticnematode to feed, grow or otherwise cause disease in a target host. Thepresent invention provides in one embodiment a method comprisingdelivery of stabilized dsRNA molecules to plant-parasitic nematodes as ameans for suppression of targeted genes in the plant-parasitic nematode,thus achieving desired control of plant disease in the nematode host.

In accomplishing the foregoing, the present invention provides a methodof inhibiting expression of a target gene in a plant-parasitic nematode,resulting in the cessation of growth, development, reproduction, and/orfeeding, and eventually may result in the death of the plant-parasiticnematode. The method comprises in one embodiment introducing partial orfully stabilized double-stranded RNA (dsRNA) nucleotide molecules,including its modified forms such as small interfering RNA (siRNA)sequences, into a nutritional composition for the plant-parasiticnematode, and making the nutritional composition or food sourceavailable to the plant-parasitic nematode. Ingestion of the nutritionalcomposition containing the double stranded or siRNA molecules results inthe uptake of the molecules by the cells of the nematode, resulting inthe inhibition of expression of at least one target gene in the cells ofthe nematode Inhibition of the target gene exerts a deleterious effectupon the nematode. The methods and associated compositions may be usedfor limiting or eliminating infection or parasitization of a plant orplant cell by a nematode, in or on any host tissue or environment inwhich a the nematode is present by providing one or more compositionscomprising the dsRNA molecules described herein in the host of thenematode.

In certain embodiments, dsRNA molecules provided by the inventioncomprise nucleotide sequences complementary to a sequence as set forthin any of SEQ ID NOs:301-1026, SEQ ID NOs:1269-1702, and SEQ IDNOs:1920-1929, the inhibition of which in a plant-parasitic nematoderesults in the reduction or removal of a protein or nucleotide sequenceagent that is essential for the nematode's growth and development orother biological function. The nucleotide sequence selected may exhibitfrom about 80% to about 100% sequence identity to one of the nucleotidesequences as set forth in SEQ ID NOs:301-1026, SEQ ID NOs:1269-1702, andSEQ ID NOs:1920-1929, including the complement thereof. However, incertain embodiments, the invention does not comprise a sequence thatexhibits 80-100% identity to SEQ ID NOs:525, 569, 797, 1293 or 1516.Such inhibition can be described as specific in that a nucleotidesequence from a portion of the target gene is chosen from which theinhibitory dsRNA or siRNA is transcribed. The method is effective ininhibiting the expression of at least one target gene and can be used toinhibit many different types of target genes in the plant-parasiticnematode.

The sequences identified as having a nematode-protective effect may bereadily expressed as dsRNA molecules through the creation of appropriateexpression constructs. For example, such sequences can be expressed as ahairpin and stem and loop structure by taking a first segmentcorresponding to a sequence selected from SEQ ID NOs:301-1026, SEQ IDNOs:1269-1702, and SEQ ID NOs:1920-1929, or a fragment thereof, linkingthis sequence to a second segment spacer region that is not homologousor complementary to the first segment, and linking this to a thirdsegment that transcribes an RNA, wherein at least a portion of the thirdsegment is substantially complementary to the first segment. Such aconstruct forms a stem and loop structure by hybridization of the firstsegment with the third segment and a loop structure forms comprising thesecond segment (WO94/01550, WO98/05770, US 2002/0048814A1, and US2003/0018993A1. dsRNA may be generated for instance in the form of adouble stranded structure such as a stem loop structure (e.g. hairpin),whereby production of siRNA targeted for a nematode sequence is enhancedby co-expression of a fragment of the targeted gene, for instance on anadditional plant expressible cassette, that leads to enhanced siRNAproduction, or reduces methylation to prevent transcriptional genesilencing of the dsRNA hairpin promoter (e.g. WO05/019408).

The methods and compositions of the present invention may be applied toany monocot and dicot plant, depending on the pathogen (e.g. nematode)control desired. Examples of such plants include, without limitation,alfalfa, artichoke, asparagus, barley, beans, beet, broccoli, cabbage,canola, carrot, cassava, cauliflower, corn, cotton, cucumber, grape,oat, onion, pea, peanut, potato, rice, rye, sorghum, soybean, spinach,squash, sugarbeet, sugarcane, sunflower, tobacco, tomato, turfgrass, andwheat plants.

Exemplary plant-parasitic nematodes from which plants may be protectedby the present invention, and their corresponding plants, include, butare not limited to: alfalfa: Ditylenchus dipsaci, Meloidogyne hapla,Meloidogyne incognita, Meloidogyne javanica, Pratylenchus spp.,Paratylenchus spp., Xiphinema spp.; banana: Radopholus similis,Helicotylenchus multicinctus, M. incognita, M. arenaria, M. javanica,Pratylenchus coffeae, Rotylenchulus reniformis; beans and peas:Meloidogyne spp., Heterodera spp., Belonolaimus spp., Helicotylenchusspp., Rotylenchulus reniformis, Paratrichodorus anemones, Trichodorusspp.; cassava: Rotylenchulus reniformis, Meloidogyne spp.; cereals:Anguina tritici (Emmer, rye, spelt wheat), Bidera avenae (oat, wheat),Ditylenchus dipsaci (rye, oat), Subanguina radicicola (oat, barley,wheat, rye), Meloidogyne naasi (barley, wheat, rye), Pratylenchus spp.(oat, wheat, barley, rye), Paratylenchus spp. (wheat), Tylenchorhynchusspp. (wheat, oat); chickpea: Heterodera cajani, Rotylenchulusreniformis, Hoplolaimus seinhorsti, Meloidogyne spp., Pratylenchus spp.;citrus: Tylenchulus semipenetrans, Radopholus similis, Radopholuscitrophilus, Hemicycliophora arenaria, Pratylenchus spp., Meloidogynespp., Bolonolaimus longicaudatus, Trichodorus spp., Paratrichodorusspp., Xiphinema spp.; clover: Meloidogyne spp., Heterodera trifolii;corn: Pratylenchus spp., Paratrichodorus minor, Longidorus spp.,Hoplolaimus columbus; cotton: Meloidogyne incognita, Belonolaimuslongicaudatus, Rotylenchulus reniformis, Hoplolaimus galeatus,Pratylenchus spp., Tylenchorhynchus spp., Paratrichodorus minor; grapes:Xiphinema spp., Pratylenchus vulnus, Meloidogyne spp., Tylenchulussemipenetrans, Rotylenchulus reniformis; grasses: Pratylenchus spp.,Longidorus spp., Paratrichodorus christiei, Xiphinema spp., Ditylenchusspp.; peanut: Pratylenchus spp., Meloidogyne hapla., Meloidogynearenaria, Criconemella spp., Belonolaimus longicaudatus; pigeon pea:Heterodera cajani, Rotylenchulus reniformis, Hoplolaimus seinhorsti,Meloidogyne spp., Pratylenchus spp.; potato: Globodera rostochiensis,Globodera pallida, Meloidogyne spp., Pratylenchus spp., Trichodorusprimitivus, Ditylenchus spp., Paratrichodorus spp., Nacobbus aberrans;rice: Aphelenchiodes besseyi, Ditylenchus angustus, Hirchmanniella spp.,Heterodera oryzae, Meloidogyne spp.; small fruits: Meloidogyne spp.;Pratylenchus spp., Xiphinema spp., Longidorus spp., Paratrichodoruschristiei, Aphelenchoides spp.; soybean: Heterodera glycines,Meloidogyne incognita, Meloidogyne javanica, Belonolaimus spp.,Hoplolaimus columbus; sugar beet: Heterodera schachtii, Ditylenchusdipsaci, Meloidogyne spp., Nacobbus aberrans, Trichodorus spp.,Longidorus spp., Paratrichodorus spp.; sugar cane: Meloidogyne spp.,Pratylenchus spp., Radopholus spp., Heterodera spp., Hoplolaimus spp.,Helicotylenchus spp., Scutellonema spp., Belonolaimus spp.,Tylenchorhynchus spp., Xiphinema spp., Longidorus spp., Paratrichodorusspp.; tobacco: Meloidogyne spp., Pratylenchus spp., Tylenchorhynchusclaytoni, Globodera tabacum, Trichodorus spp., Xiphinema americanum,Ditylenchus dipsaci, Paratrichodorus spp.; and tomato: Pratylenchusspp., Meloidogyne spp.

The invention also provides combinations of methods and compositions forcontrolling infection by plant-parasitic nematodes. One means provides adsRNA method as described herein for protecting plants fromplant-parasitic nematodes along with one or more chemical agents thatexhibit features different from those exhibited by the dsRNA methods andcompositions, and can interfere with nematode growth or development.

A. Nucleic Acid Compositions and Constructs

The invention provides recombinant DNA constructs for use in achievingstable transformation of particular host targets. Transformed hosttargets may express effective levels of preferred dsRNA or siRNAmolecules from the recombinant DNA constructs. Pairs of isolated andpurified nucleotide sequences may be provided from cDNA library and/orgenomic library information. The pairs of nucleotide sequences may bederived from any nematode for use as thermal amplification primers togenerate the dsRNA and siRNA molecules of the present invention.

As used herein, the term “nucleic acid” refers to a single ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesread from the 5′ to the 3′ end. The “nucleic acid” may also optionallycontain non-naturally occurring or altered nucleotide bases that permitcorrect read through by a polymerase and do not reduce expression of apolypeptide encoded by that nucleic acid. The term “nucleotide sequence”or “nucleic acid sequence” refers to both the sense and antisensestrands of a nucleic acid as either individual single strands or in theduplex. The term “ribonucleic acid” (RNA) is inclusive of RNAi(inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interferingRNA), mRNA (messenger RNA), miRNA (micro-RNA), tRNA (transfer RNA,whether charged or discharged with a corresponding acylated amino acid),and cRNA (complementary RNA) and the term “deoxyribonucleic acid” (DNA)is inclusive of cDNA and genomic DNA and DNA-RNA hybrids. The words“nucleic acid segment”, “nucleotide sequence segment”, or more generally“segment” will be understood by those in the art as a functional termthat includes both genomic sequences, ribosomal RNA sequences, transferRNA sequences, messenger RNA sequences, operon sequences and smallerengineered nucleotide sequences that express or may be adapted toexpress, proteins, polypeptides or peptides.

Provided according to the invention are nucleotide sequences, theexpression of which results in an RNA sequence which is substantiallyhomologous to all or part of an RNA molecule of a targeted gene in aplant-parasitic nematode that comprises an RNA sequence encoded by anucleotide sequence within the genome of the nematode. Thus, afteringestion of the stabilized RNA sequence down-regulation of thenucleotide sequence of the target gene in the cells of theplant-parasitic nematode may be obtained resulting in a deleteriouseffect on the growth, viability, proliferation, or reproduction of thenematode.

As used herein, the term “substantially homologous” or “substantialhomology”, with reference to a nucleic acid sequence, includes anucleotide sequence that hybridizes under stringent conditions to thecoding sequence of any of SEQ ID NOs:301-1026, SEQ ID NOs:1269-1702, andSEQ ID NOs:1920-1929, as set forth in the sequence listing, or thecomplements thereof. Sequences that hybridize under stringent conditionsto any of SEQ ID NOs:301-1026, SEQ ID NOs:1269-1702, and SEQ IDNOs:1920-1929, or the complements thereof, are those that allow anantiparallel alignment to take place between the two sequences, and thetwo sequences are then able, under stringent conditions, to formhydrogen bonds with corresponding bases on the opposite strand to form aduplex molecule that is sufficiently stable under the stringentconditions to be detectable using methods well known in the art.Substantially homologous sequences have preferably from about 70% toabout 80% sequence identity, or more preferably from about 80% to about85% sequence identity, or most preferable from about 90% to about 95%sequence identity, to about 99% sequence identity, to the referentnucleotide sequences as set forth in any of SEQ ID NOs:301-1026, SEQ IDNOs:1269-1702, and SEQ ID NOs:1920-1929, in the sequence listing, or thecomplements thereof.

As used herein, the term “ortholog” refers to a gene in two or morespecies that has evolved from a common ancestral nucleotide sequence,and may retain the same function in the two or more species.

As used herein, the term “sequence identity”, “sequence similarity” or“homology” is used to describe sequence relationships between two ormore nucleotide sequences. The percentage of “sequence identity” betweentwo sequences is determined by comparing two optimally aligned sequencesover a comparison window, wherein the portion of the sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparison,and multiplying the result by 100 to yield the percentage of sequenceidentity. A sequence that is identical at every position in comparisonto a reference sequence is said to be identical to the referencesequence and vice-versa. A first nucleotide sequence when observed inthe 5′ to 3′ direction is said to be a “complement” of, or complementaryto, a second or reference nucleotide sequence observed in the 3′ to 5′direction if the first nucleotide sequence exhibits completecomplementarity with the second or reference sequence. As used herein,nucleic acid sequence molecules are said to exhibit “completecomplementarity” when every nucleotide of one of the sequences read 5′to 3′ is complementary to every nucleotide of the other sequence whenread 3′ to 5′. A nucleotide sequence that is complementary to areference nucleotide sequence will exhibit a sequence identical to thereverse complement sequence of the reference nucleotide sequence. Theseterms and descriptions are well defined in the art and are easilyunderstood by those of ordinary skill in the art.

As used herein, a “comparison window” refers to a conceptual segment ofat least 6 contiguous positions, usually about 50 to about 100, moreusually about 100 to about 150, in which a sequence is compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. The comparison window may compriseadditions or deletions (i.e. gaps) of about 20% or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. Those skilled in the artshould refer to the detailed methods used for sequence alignment in theWisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Drive Madison, Wis., USA) or refer to Ausubel et al.(1998) for a detailed discussion of sequence analysis.

The present invention provides DNA sequences capable of being expressedas an RNA in a cell or microorganism to inhibit target gene expressionin a cell, tissue or organ of a plant-parasitic nematode. The sequencescomprise a DNA molecule coding for one or more different nucleotidesequences, wherein each of the different nucleotide sequences comprisesa sense nucleotide sequence and an antisense nucleotide sequence. Thesequences may be connected by a spacer sequence coding for a dsRNAmolecule of the present invention. The spacer sequence can constitutepart of the sense nucleotide sequence or the antisense nucleotidesequence and forms within the dsRNA molecule between the sense andantisense sequences. The sense nucleotide sequence or the antisensenucleotide sequence is substantially identical to the nucleotidesequence of the target gene or a derivative thereof or a complementarysequence thereto. The dsDNA molecule may be placed operably under thecontrol of a promoter sequence that functions in the cell, tissue ororgan of the host expressing the dsDNA to produce dsRNA molecules. Inone embodiment, the DNA sequence may be derived from a nucleotidesequence as set forth in SEQ ID NOs:301-1026, SEQ ID NOs:1269-1702, andSEQ ID NOs:1920-1929, in the sequence listing.

The invention also provides a DNA sequence for expression in a cell of aplant that, upon expression of the DNA to RNA and ingestion by aplant-parasitic nematode achieves suppression of a target gene in acell, tissue or organ of a plant-parasitic nematode. The dsRNA maycomprise one or multiple structural gene sequences, wherein each of thestructural gene sequences comprises a sense nucleotide sequence and anantisense nucleotide sequence that may be connected by a spacer sequencethat forms a loop within the complementary and antisense sequences. Thesense nucleotide sequence or the antisense nucleotide sequence issubstantially identical to the nucleotide sequence of the target gene,derivative thereof, or sequence complementary thereto. The one or morestructural gene sequences may be placed operably under the control ofone or more promoter sequences, at least one of which is operable in thecell, tissue or organ of a prokaryotic or eukaryotic organism,particularly a plant cell. Methods to express a gene suppressionmolecule in plants are known (e.g. WO06073727 A2; US Publication2006/0200878 A1), and may be used to express a nucleotide sequence ofthe present invention.

A gene sequence or fragment for plant-parasitic nematode controlaccording to the invention may be cloned between two tissue specificpromoters, such as two root specific promoters which are operable in atransgenic plant cell and therein expressed to produce mRNA in thetransgenic plant cell that form dsRNA molecules thereto. Examples ofroot specific promoters are known in the art (e.g. the nematode-inducedRB7 promoter; U.S. Pat. No. 5,459,252; Opperman et al. 1994). The dsRNAmolecules contained in plant tissues are ingested by a plant-parasiticnematode so that the intended suppression of the target gene expressionis achieved.

The cauliflower mosaic virus 35S promoter, an archetypal strong promotercommon in transgenic plant applications, or a related promoter such asthe E35S or the FMV promoter, may be employed for driving nematoderesistance genes, particularly for cyst nematodes (see Example 8).Promoters have also been identified that direct gene expression atnematode-induced feeding structures within a plant (e.g. Gheysen andFenoll, 2002). Thus, a promoter identified from among genes that arereproducibly expressed in feeding sites may be utilized. Examples ofgenes up-regulated in feeding sites (syncytia) formed by nematodesinclude Hs1pro-1 (Thurau et al. 2003), AtSUC2 normally expressed incompanion cells (Juergensen et al. 2003), At17.1 expressed in vasculartissues and root tips (Mazarei et al. 2004), FGAM synthase(phosphoribosylformyl-glycinamidine synthase) (Vaghchhipawala et al.2004), and ABI3 (De Meutter et al. 2005), among others. Syncytia formedin response to cyst nematodes have been described as symplasticallyisolated lacking plasmodesmata to surrounding tissues (Bockenhoff andGrundler 1994; Bockenhoff et al. 1996), however, it has been shown thatmacromolecules up to 30 kD can move from phloem companion cells into thesyncytium (Hoth et al. 2005). Therefore, gene expression in the phloemmay also be suited for delivery of effector molecules into feedingsites.

A nucleotide sequence provided by the present invention may comprise aninverted repeat separated by a “spacer sequence.” The spacer sequencemay be a region comprising any sequence of nucleotides that facilitatessecondary structure formation between each repeat, where this isrequired. In one embodiment of the present invention, the spacersequence is part of the sense or antisense coding sequence for mRNA. Thespacer sequence may alternatively comprise any combination ofnucleotides or homologues thereof that are capable of being linkedcovalently to a nucleic acid molecule. The spacer sequence may comprise,for example, a sequence of nucleotides of at least about 10-100nucleotides in length, or alternatively at least about 100-200nucleotides in length, at least 200-400 about nucleotides in length, orat least about 400-500 nucleotides in length.

The nucleic acid molecules or fragments of the nucleic acid molecules orother nucleic acid molecules in the sequence listing are capable ofspecifically hybridizing to other nucleic acid molecules under certaincircumstances. As used herein, two nucleic acid molecules are said to becapable of specifically hybridizing to one another if the two moleculesare capable of forming an anti-parallel, double-stranded nucleic acidstructure. A nucleic acid molecule is said to be the complement ofanother nucleic acid molecule if they exhibit complete complementarity.Two molecules are said to be “minimally complementary” if they canhybridize to one another with sufficient stability to permit them toremain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to becomplementary if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook, et al. (1989), and by Haymes etal. (1985).

Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. Thus, in order for anucleic acid molecule or a fragment of the nucleic acid molecule toserve as a primer or probe it needs only be sufficiently complementaryin sequence to be able to form a stable double-stranded structure underthe particular solvent and salt concentrations employed.

Appropriate stringency conditions which promote DNA hybridization are,for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology(1989). For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged. A nucleic acid for use in the present invention mayspecifically hybridize to one or more of nucleic acid molecules from anematode or complements thereof under such conditions. Preferably, anucleic acid for use in the present invention will exhibit at least fromabout 80%, or at least from about 90%, or at least from about 95%, or atleast from about 98% or even about 100% sequence identity with one ormore nucleic acid molecules as set forth in SEQ ID NOs:301-1026, SEQ IDNOs:1269-1702, and SEQ ID NOs:1920-1929, in the sequence listing.

Nucleic acids of the present invention may also be synthesized, eithercompletely or in part, especially where it is desirable to provideplant-preferred sequences, by methods known in the art. Thus, all or aportion of the nucleic acids of the present invention may be synthesizedusing codons preferred by a selected host. Species-preferred codons maybe determined, for example, from the codons used most frequently in theproteins expressed in a particular host species. Other modifications ofthe nucleotide sequences may result in mutants having slightly alteredactivity.

dsRNA or siRNA nucleotide sequences comprise double strands ofpolymerized ribonucleotide and may include modifications to either thephosphate-sugar backbone or the nucleoside. Modifications in RNAstructure may be tailored to allow specific genetic inhibition. In oneembodiment, the dsRNA molecules may be modified through an enzymaticprocess so that siRNA molecules may be generated. The siRNA canefficiently mediate the down-regulation effect for some target genes insome pathogens. This enzymatic process may be accomplished by utilizingan RNAse III enzyme or a DICER enzyme, present in the cells of aninsect, a vertebrate animal, a fungus or a plant in the eukaryotic RNAipathway (Elbashir et al., 2001; Hamilton and Baulcombe, 1999). Thisprocess may also utilize a recombinant DICER or RNAse III introducedinto the cells of a target insect through recombinant DNA techniquesthat are readily known to the skilled in the art. Both the DICER enzymeand RNAse III, being naturally occurring in a pathogen or being madethrough recombinant DNA techniques, cleave larger dsRNA strands intosmaller oligonucleotides. The DICER enzymes specifically cut the dsRNAmolecules into siRNA pieces each of which is about 19-25 nucleotides inlength while the RNAse III enzymes normally cleave the dsRNA moleculesinto 12-15 base-pair siRNA. The siRNA molecules produced by the eitherof the enzymes have 2 to 3 nucleotide 3′ overhangs, and 5′ phosphate and3′ hydroxyl termini. The siRNA molecules generated by RNAse III enzymeare the same as those produced by Dicer enzymes in the eukaryotic RNAipathway and are hence then targeted and degraded by an inherent cellularRNA-degrading mechanism after they are subsequently unwound, separatedinto single-stranded RNA and hybridize with the RNA sequencestranscribed by the target gene. This process results in the effectivedegradation or removal of the RNA sequence encoded by the nucleotidesequence of the target gene in the pathogen. The outcome is thesilencing of a particularly targeted nucleotide sequence within thepathogen. Detailed descriptions of enzymatic processes can be found inHannon (2002).

A nucleotide sequence of the present invention can be recorded oncomputer readable media. As used herein, “computer readable media”refers to any tangible medium of expression that can be read andaccessed directly by a computer. Such media include, but are not limitedto: magnetic storage media, such as floppy discs, hard disc, storagemedium, and magnetic tape: optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; optical characterrecognition formatted computer files, and hybrids of these categoriessuch as magnetic/optical storage media. A skilled artisan can readilyappreciate that any of the presently known computer readable mediums canbe used to create a manufacture comprising computer readable mediumhaving recorded thereon a nucleotide sequence of the present invention.

As used herein, “recorded” refers to a process for storing informationon computer readable medium. A skilled artisan can readily adopt any ofthe presently known methods for recording information on computerreadable medium to generate media comprising the nucleotide sequenceinformation of the present invention. A variety of data storagestructures are available to a skilled artisan for creating a computerreadable medium having recorded thereon a nucleotide sequence of thepresent invention. The choice of the data storage structure willgenerally be based on the means chosen to access the stored information.In addition, a variety of data processor programs and formats can beused to store the nucleotide sequence information of the presentinvention on computer readable medium. The sequence information can berepresented in a word processing text file, formatted incommercially-available software such as WordPerfect and Microsoft Word,or represented in the form of an ASCII text file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like. The skilledartisan can readily adapt any number of data processor structuringformats (e.g. text file or database) in order to obtain computerreadable medium having recorded thereon the nucleotide sequenceinformation of the present invention.

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable medium.Software that implements the BLAST (Altschul et al., 1990) and BLAZE(Brutlag, et al., 1993) search algorithms on a Sybase system can be usedto identify open reading frames (ORFs) within sequences such as theUnigenes and EST's that are provided herein and that contain homology toORFs or proteins from other organisms. Such ORFs are protein-encodingfragments within the sequences of the present invention and are usefulin producing commercially important proteins such as enzymes used inamino acid biosynthesis, metabolism, transcription, translation, RNAprocessing, nucleic acid and a protein degradation, proteinmodification, and DNA replication, restriction, modification,recombination, and repair.

The present invention further provides systems, particularlycomputer-based systems, which contain the sequence information describedherein. Such systems are designed to identify commercially importantfragments of the nucleic acid molecule of the present invention. As usedherein, “a computer-based system” refers to the hardware means, softwaremeans, and data storage means used to analyze the nucleotide sequenceinformation of the present invention. The minimum hardware means of thecomputer-based systems of the present invention comprises a centralprocessing unit (CPU), input means, output means, and data storagemeans. A skilled artisan can readily appreciate that any one of thecurrently available computer-based system are suitable for use in thepresent invention.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequences or sequence(s) are chosen based on a three-dimensionalconfiguration that is formed upon the folding of the target motif. Thereare a variety of target motifs known in the art. Protein target motifsinclude, but are not limited to, enzymatic active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, cis elements, hairpin structures and inducibleexpression elements (protein binding sequences).

B. Recombinant Vectors and Host Cell Transformation

A recombinant DNA vector may, for example, be a linear or a closedcircular plasmid. The vector system may be a single vector or plasmid ortwo or more vectors or plasmids that together contain the total DNA tobe introduced into the genome of the bacterial host. In addition, abacterial vector may be an expression vector. Nucleic acid molecules asset forth in SEQ ID NOs:301-1026 SEQ ID NOs:1269-1702, and SEQ IDNOs:1920-1929, or fragments thereof, can, for example, be suitablyinserted into a vector under the control of a suitable promoter thatfunctions in one or more microbial hosts to drive expression of a linkedcoding sequence or other DNA sequence. Many vectors are available forthis purpose, and selection of the appropriate vector will depend mainlyon the size of the nucleic acid to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components depending on its function (amplification ofDNA or expression of DNA) and the particular host cell with which it iscompatible. The vector components for bacterial transformation generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more selectable marker genes,and an inducible promoter allowing the expression of exogenous DNA.

Expression and cloning vectors generally contain a selection gene, alsoreferred to as a selectable marker. This gene encodes a proteinnecessary for the survival or growth of transformed host cells grown ina selective culture medium. Typical selection genes encode proteins that(a) confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.Those cells that are successfully transformed with a heterologousprotein or fragment thereof produce a protein conferring drug resistanceand thus survive the selection regimen.

An expression vector for producing a mRNA can also contain an induciblepromoter that is recognized by a host bacterial organism and is operablylinked to the nucleic acid. Inducible promoters suitable for use withbacterial hosts include β-lactamase promoter, E. coli λ phage PL and PRpromoters, and E. coli galactose promoter, arabinose promoter, alkalinephosphatase promoter, tryptophan (trp) promoter, and the lactose operonpromoter and variations thereof and hybrid promoters such as the tacpromoter. However, other known bacterial inducible promoters aresuitable.

The term “operably linked”, as used in reference to a regulatorysequence and a structural nucleotide sequence, means that the regulatorysequence causes regulated expression of the linked structural nucleotidesequence. “Regulatory sequences” or “control elements” refer tonucleotide sequences located upstream (5′ noncoding sequences), within,or downstream (3′ non-translated sequences) of a structural nucleotidesequence, and which influence the timing and level or amount oftranscription, RNA processing or stability, or translation of theassociated structural nucleotide sequence. Regulatory sequences mayinclude promoters, translation leader sequences, introns, enhancers,stem-loop structures, repressor binding sequences, and polyadenylationrecognition sequences and the like.

Construction of suitable vectors containing one or more of theabove-listed components employs standard recombinant DNA techniques.Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligatedin the form desired to generate the plasmids required. Examples ofavailable bacterial expression vectors include, but are not limited to,the multifunctional E. coli cloning and expression vectors such asBluescript™ (Stratagene, La Jolla, Calif.), in which, for example, anucleic acid, or fragment thereof, shown in FIG. 3 may be ligated intothe vector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of β-galactosidase so that a hybrid protein isproduced; pIN vectors (Van Heeke and Schuster, 1989); and the like.

The present invention also contemplates transformation of a nucleotidesequence of the present invention into a plant to achievenematode-inhibitory levels of expression of one or more dsRNA molecules.A transformation vector can be readily prepared using methods availablein the art. The transformation vector comprises one or more nucleotidesequences that is/are capable of being transcribed to an RNA moleculeand that is/are substantially homologous and/or complementary to one ormore nucleotide sequences encoded by the genome of the target nematode,such that upon uptake of the RNA transcribed from the one or morenucleotide sequences by the target plant-parasitic nematode, there isdown-regulation of expression of at least one of the respectivenucleotide sequences of the genome of the nematode.

The transformation vector may be termed a dsDNA construct and may alsobe defined as a recombinant molecule, a disease control agent, a geneticmolecule or a chimeric genetic construct. A chimeric genetic constructof the present invention may comprise, for example, nucleotide sequencesencoding one or more antisense transcripts, one or more sensetranscripts, one or more of each of the aforementioned, wherein all orpart of a transcript there from is homologous to all or part of an RNAmolecule comprising an RNA sequence encoded by a nucleotide sequencewithin the genome of a pathogen.

In one embodiment a plant transformation vector comprises an isolatedand purified DNA molecule comprising a heterologous promoter operativelylinked to one or more nucleotide sequences of the present invention. Thenucleotide sequence is selected from the group consisting of SEQ IDNOs:301-1026, SEQ ID NOs:1269-1702, and SEQ ID NOs:1920-1929, as setforth in the sequence listing. The nucleotide sequence includes asegment coding all or part of an RNA present within a targeted nematodeRNA transcript and may comprise inverted repeats of all or a part of atargeted nematode RNA. The DNA molecule comprising the expression vectormay also contain a functional intron sequence positioned either upstreamof the coding sequence or even within the coding sequence, and may alsocontain a five prime (5′) untranslated leader sequence (i.e., a UTR or5′-UTR) positioned between the promoter and the point of translationinitiation.

A plant transformation vector may contain sequences from more than onegene, thus allowing production of more than one dsRNA for inhibitingexpression of two or more genes in cells of a target nematode. Oneskilled in the art will readily appreciate that segments of DNA whosesequence corresponds to that present in different genes can be combinedinto a single composite DNA segment for expression in a transgenicplant. Alternatively, a plasmid of the present invention alreadycontaining at least one DNA segment can be modified by the sequentialinsertion of additional DNA segments between the enhancer and promoterand terminator sequences. In the disease control agent of the presentinvention designed for the inhibition of multiple genes, the genes to beinhibited can be obtained from the same plant-parasitic nematode speciesin order to enhance the effectiveness of the control agent. In certainembodiments, the genes can be derived from different plant-parasiticnematodes in order to broaden the range of nematodes against which theagent(s) is/are effective. When multiple genes are targeted forsuppression or a combination of expression and suppression, apolycistronic DNA element can be fabricated as illustrated and disclosedin US Publication No. US 2004-0029283.

Promoters that function in different plant species are also well knownin the art. Promoters useful for expression of polypeptides in plantsinclude those that are inducible, viral, synthetic, or constitutive asdescribed in Odell et al. (1985), and/or promoters that are temporallyregulated, spatially regulated, and spatio-temporally regulated.Preferred promoters include the enhanced CaMV35S promoters, and theFMV35S promoter. A fragment of the CaMV35S promoter exhibitingroot-specificity may also be preferred. For the purpose of the presentinvention, it may be preferable to achieve the highest levels ofexpression of these genes within the root tissues of plants. A number ofroot-specific promoters have been identified and are known in the art(e.g. U.S. Pat. Nos. 5,110,732; 5,837,848; 5,459,252; Hirel et al.1992).

A recombinant DNA vector or construct of the present invention maycomprise a selectable marker that confers a selectable phenotype onplant cells. Selectable markers may also be used to select for plants orplant cells that contain the exogenous nucleic acids encodingpolypeptides or proteins of the present invention. The marker may encodebiocide resistance, antibiotic resistance (e.g., kanamycin, G418bleomycin, hygromycin, etc.), or herbicide resistance (e.g., glyphosate,etc.). Examples of selectable markers include, but are not limited to, aneo gene which codes for kanamycin resistance and can be selected forusing kanamycin, G418, etc., a bar gene which codes for bialaphosresistance; a mutant EPSP synthase gene which encodes glyphosateresistance; a nitrilase gene which confers resistance to bromoxynil; amutant acetolactate synthase gene (ALS) which confers imidazolinone orsulfonylurea resistance; and a methotrexate resistant DHFR gene.Multiple selectable markers are available that confer resistance toampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin,spectinomycin, rifampicin, and tetracycline, and the like. Examples ofsuch selectable markers are illustrated in U.S. Pat. Nos. 5,550,318;5,633,435; 5,780,708 and 6,118,047.

A recombinant vector or construct of the present invention may alsoinclude a screenable marker. Screenable markers may be used to monitorexpression. Exemplary screenable markers include a β-glucuronidase oruidA gene (GUS) which encodes an enzyme for which various chromogenicsubstrates are known (Jefferson et al., 1987); an R-locus gene, whichencodes a product that regulates the production of anthocyanin pigments(red color) in plant tissues (Dellaporta et al., 1988); a β-lactamasegene (Sutcliffe et al., 1978), a gene which encodes an enzyme for whichvarious chromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a luciferase gene (Ow et al., 1986) a xylE gene(Zukowski et al., 1983) which encodes a catechol dioxygenase that canconvert chromogenic catechols; an α-amylase gene (Ikatu et al., 1990); atyrosinase gene (Katz et al., 1983) which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone which in turn condenses tomelanin; an α-galactosidase, which catalyzes a chromogenic α-galactosesubstrate.

Preferred plant transformation vectors include those derived from a Tiplasmid of Agrobacterium tumefaciens (e.g. U.S. Pat. Nos. 4,536,475,4,693,977, 4,886,937, 5,501,967 and EP 0 122 791). Agrobacteriumrhizogenes plasmids (or “Ri”) are also useful and known in the art.Other preferred plant transformation vectors include those disclosed,e.g., by Herrera-Estrella (1983); Bevan (1983), Klee (1985) and EP 0 120516.

In general it may be preferred to introduce a functional recombinant DNAat a non-specific location in a plant genome. In special cases it may beuseful to insert a recombinant DNA construct by site-specificintegration. Several site-specific recombination systems exist which areknown to function implants include cre-lox as disclosed in U.S. Pat. No.4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695.

Suitable methods for transformation of host cells for use with thecurrent invention are believed to include virtually any method by whichDNA can be introduced into a cell (see, for example, Miki et al., 1993),such as by transformation of protoplasts (U.S. Pat. No. 5,508,184;Omirulleh et al., 1993), by desiccation/inhibition-mediated DNA uptake(Potrykus et al., 1985), by electroporation (U.S. Pat. No. 5,384,253),by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S.Pat. No. 5,302,523; and U.S. Pat. No. 5,464,765), byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055;5,591,616; 5,693,512; 5,824,877; 5,981,840; 6,384,301) and byacceleration of DNA coated particles (U.S. Pat. Nos. 5,015,580;5,550,318; 5,538,880; 6,160,208; 6,399,861; 6,403,865; Padgette et al.1995), etc. Through the application of techniques such as these, thecells of virtually any species may be stably transformed. In the case ofmulticellular species, the transgenic cells may be regenerated intotransgenic organisms.

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium (for example, Horsch et al., 1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. Descriptions of Agrobacterium vectorsystems and methods for Agrobacterium-mediated gene transfer areprovided by numerous references, including Gruber et al. 1993; Miki etal., 1993, Moloney et al., 1989, and U.S. Pat. Nos. 4,940,838 and5,464,763. Other bacteria such as Sinorhizobium, Rhizobium, andMesorhizobium that interact with plants naturally can be modified tomediate gene transfer to a number of diverse plants. Theseplant-associated symbiotic bacteria can be made competent for genetransfer by acquisition of both a disarmed Ti plasmid and a suitablebinary vector (Broothaerts et al. 2005).

Methods for the creation of transgenic plants and expression ofheterologous nucleic acids in plants in particular are known and may beused with the nucleic acids provided herein to prepare transgenic plantsthat exhibit reduced susceptibility to feeding by a target nematode.Plant transformation vectors can be prepared, for example, by insertingthe dsRNA producing nucleic acids disclosed herein into planttransformation vectors and introducing these into plants. One knownvector system has been derived by modifying the natural gene transfersystem of Agrobacterium tumefaciens. The natural system comprises largeTi (tumor-inducing)-plasmids containing a large segment, known as T-DNA,which is transferred to transformed plants. Another segment of the Tiplasmid, the vir region, is responsible for T-DNA transfer. The T-DNAregion is bordered by terminal repeats. In the modified binary vectorsthe tumor-inducing genes have been deleted and the functions of the virregion are utilized to transfer foreign DNA bordered by the T-DNA bordersequences. The T-region may also contain a selectable marker forefficient recovery of transgenic plants and cells, and a multiplecloning site for inserting sequences for transfer such as a dsRNAencoding nucleic acid.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single simple recombinant DNA sequence insertedinto one chromosome and is referred to as a transgenic event. Suchtransgenic plants can be referred to as being heterozygous for theinserted exogenous sequence. A transgenic plant homozygous with respectto a transgene can be obtained by sexually mating (selfing) anindependent segregant transgenic plant that contains a single exogenousgene sequence to itself, for example an F0 plant, to produce F1 seed.One fourth of the F1 seed produced will be homozygous with respect tothe transgene. Germinating F1 seed results in plants that can be testedfor heterozygosity, typically using a SNP assay or a thermalamplification assay that allows for the distinction betweenheterozygotes and homozygotes (i.e., a zygosity assay). Crossing aheterozygous plant with itself or another heterozygous plant results inonly heterozygous progeny.

C. Nucleic Acid Expression and Target Gene Suppression

The present invention provides, as an example, a transformed host plantof a pathogenic target organism, transformed plant cells and transformedplants and their progeny. The transformed plant cells and transformedplants may be engineered to express one or more of the dsRNA or siRNAsequences, under the control of a heterologous promoter, describedherein to provide a pathogen-protective effect. These sequences may beused for gene suppression in a pathogen, thereby reducing the level orincidence of disease caused by the pathogen on a protected transformedhost organism. As used herein the words “gene suppression” are intendedto refer to any of the well-known methods for reducing the levels ofprotein produced as a result of gene transcription to mRNA andsubsequent translation of the mRNA.

Gene suppression is also intended to mean the reduction of proteinexpression from a gene or a coding sequence includingposttranscriptional gene suppression and transcriptional suppression.Posttranscriptional gene suppression is mediated by the homology betweenof all or a part of a mRNA transcribed from a gene or coding sequencetargeted for suppression and the corresponding double stranded RNA usedfor suppression, and refers to the substantial and measurable reductionof the amount of available mRNA available in the cell for binding byribosomes. The transcribed RNA can be in the sense orientation to effectwhat is called co-suppression, in the anti-sense orientation to effectwhat is called anti-sense suppression, or in both orientations producinga dsRNA to effect what is called RNA interference (RNAi).

Transcriptional suppression is mediated by the presence in the cell of adsRNA gene suppression agent exhibiting substantial sequence identity toa promoter DNA sequence or the complement thereof to effect what isreferred to as promoter trans suppression. Gene suppression may beeffective against a native plant gene associated with a trait, e.g., toprovide plants with reduced levels of a protein encoded by the nativegene or with enhanced or reduced levels of an affected metabolite. Genesuppression can also be effective against target genes in aplant-parasitic nematode that may ingest or contact plant materialcontaining gene suppression agents, specifically designed to inhibit orsuppress the expression of one or more homologous or complementarysequences in the cells of the nematode. Post-transcriptional genesuppression by anti-sense or sense oriented RNA to regulate geneexpression in plant cells is disclosed in U.S. Pat. Nos. 5,107,065,5,759,829, 5,283,184, and 5,231,020. The use of dsRNA to suppress genesin plants is disclosed in WO 99/53050, WO 99/49029, U.S. Publication No.2003/0175965, and 2003/0061626, U.S. patent application Ser. No.10/465,800, and U.S. Pat. Nos. 6,506,559, and 6,326,193.

A beneficial method of post transcriptional gene suppression versus aplant-parasitic nematode employs both sense-oriented andanti-sense-oriented, transcribed RNA which is stabilized, e.g., as ahairpin and stem and loop structure. A preferred DNA construct foreffecting post transcriptional gene suppression is one in which a firstsegment encodes an RNA exhibiting an anti-sense orientation exhibitingsubstantial identity to a segment of a gene targeted for suppression,which is linked to a second segment encoding an RNA exhibitingsubstantial complementarity to the first segment. Such a construct formsa stem and loop structure by hybridization of the first segment with thesecond segment and a loop structure from the nucleotide sequenceslinking the two segments (see WO94/01550, WO98/05770, US 2002/0048814,and US 2003/0018993). Co-expression with an additional target genesegment may also be employed, as noted above (e.g. WO05/019408).

According to one embodiment of the present invention, there is provideda nucleotide sequence, for which in vitro expression results intranscription of a stabilized RNA sequence that is substantiallyhomologous to an RNA molecule of a targeted gene in a plant-parasiticnematode that comprises an RNA sequence encoded by a nucleotide sequencewithin the genome of the nematode. Thus, after the plant-parasiticnematode ingests the stabilized RNA sequence, a down-regulation of thenucleotide sequence corresponding to the target gene in the cells of atarget nematode is effected.

In certain embodiments of the invention, expression of a fragment of atleast 21 contiguous nucleotides of a nucleic acid sequence of any of SEQID NOs:301-1026, SEQ ID NOs:1269-1702, and SEQ ID NOs:1920-1929 may beutilized, including expression of a fragment of up to 21, 36, 60, 100,550, or 1000 contiguous nucleotides, or sequences displaying 90-100%identity with such sequences, or their complements. In specificembodiments, a nucleotide provided by the invention may comprise asequence selected from the group described in Table 4, including alocation on such sequence spanning nucleotides as described in Table 4.In yet other embodiments, a nucleotide provided by the invention may bedescribed as comprising one or more of nucleotides 1-21, 22-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500,501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900,901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200,1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 23-75,76-125, 126-175, 176-225, 226-275, 276-325, 326-375, 376-425, 426-475,476-525, 526-575, 576-625, 626-675, 676-725, 726-775, 776-825, 826-875,876-925, 926-975, 976-1025, 1026-1075, 1076-1125, 1126-1175, 1176-1225,1226-1275, 1276-1325, 1326-1375, 1376-1425, 1426-1475, 1476-1525,1526-1575, 1576-1625, 1626-1675, 1676-1725, 1726-1775, 1776-1825,1826-1875, 1876-1925, 1926-1975, 1976-2025, 2026-2075, 2076-2125, 1-550,200-750, 300-850, 400-950, 500-1050, 600-1150, 700-1250, 800-1350,900-1450, 1000-1550, 1100-1650, 1200-1750, 1300-1850, 1400-1950,1500-2050, up to the full length of the sequence, of one or more of SEQID NOs:301-1026, SEQ ID NOs:1269-1702, and SEQ ID NOs:1920-1929.However, in certain embodiments, the invention does not comprise SEQ IDNOs:525, 569, 797, 1293 or 1516, or fragments thereof. Methods forselecting specific sub-sequences as targets for siRNA-mediated genesuppression are known in the art (e.g. Reynolds et al. 2004).

Inhibition of a target gene using the stabilized dsRNA technology of thepresent invention is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition. RNA containing a nucleotide sequences identical to a portionof the target gene is preferred for inhibition. RNA sequences withinsertions, deletions, and single point mutations relative to the targetsequence have also been found to be effective for inhibition. Inperformance of the present invention, it is preferred that theinhibitory dsRNA and the portion of the target gene share at least fromabout 80% sequence identity, or from about 90% sequence identity, orfrom about 95% sequence identity, or from about 99% sequence identity,or even about 100% sequence identity. Alternatively, the duplex regionof the RNA may be defined functionally as a nucleotide sequence that iscapable of hybridizing with a portion of the target gene transcript. Aless than full length sequence exhibiting a greater homology compensatesfor a longer less homologous sequence. The length of the identicalnucleotide sequences may be at least about 25, 50, 100, 200, 300, 400,500 or at least about 1000 bases. Normally, a sequence of greater than20-100 nucleotides should be used, though a sequence of greater thanabout 200-300 nucleotides would be preferred, and a sequence of greaterthan about 500-1000 nucleotides would be especially preferred dependingon the size of the target gene. The invention has the advantage of beingable to tolerate sequence variations that might be expected due togenetic mutation, strain polymorphism, or evolutionary divergence. Theintroduced nucleic acid molecule may not need to be absolute homology,may not need to be full length, relative to either the primarytranscription product or fully processed mRNA of the target gene.Therefore, those skilled in the art need to realize that, as disclosedherein, 100% sequence identity between the RNA and the target gene isnot required to practice the present invention.

Inhibition of target gene expression may be quantified by measuringeither the endogenous target RNA or the protein produced by translationof the target RNA and the consequences of inhibition can be confirmed byexamination of the outward properties of the cell or organism.Techniques for quantifying RNA and proteins are well known to one ofordinary skill in the art.

In certain embodiments gene expression is inhibited by at least 10%,preferably by at least 33%, more preferably by at least 50%, and yetmore preferably by at least 80%. In particularly preferred embodimentsof the invention gene expression is inhibited by at least 80%, morepreferably by at least 90%, more preferably by at least 95%, or by atleast 99% within cells in the pathogen so a significant inhibition takesplace. Significant inhibition is intended to refer to sufficientinhibition that results in a detectable phenotype (e.g., cessation ofgrowth, feeding, development, mortality, etc.) or a detectable decreasein RNA and/or protein corresponding to the target gene being inhibited.Although in certain embodiments of the invention inhibition occurs insubstantially all cells of the plant-parasitic nematode, in otherpreferred embodiments inhibition occurs in only a subset of cellsexpressing the gene.

dsRNA molecules may be synthesized either in vivo or in vitro. The dsRNAmay be formed by a single self-complementary RNA strand or from twocomplementary RNA strands. Endogenous RNA polymerase of the cell maymediate transcription in vivo, or cloned RNA polymerase can be used fortranscription in vivo or in vitro Inhibition may be targeted by specifictranscription in an organ, tissue, or cell type; stimulation of anenvironmental condition (e.g., infection, stress, temperature, chemicalinducers); and/or engineering transcription at a developmental stage orage. The RNA strands may or may not be polyadenylated; the RNA strandsmay or may not be capable of being translated into a polypeptide by acell's translational apparatus.

A RNA, dsRNA, siRNA, or miRNA of the present invention may be producedchemically or enzymatically by one skilled in the art through manual orautomated reactions or in vivo in another organism. RNA may also beproduced by partial or total organic synthesis; any modifiedribonucleotide can be introduced by in vitro enzymatic or organicsynthesis. The RNA may be synthesized by a cellular RNA polymerase or abacteriophage RNA polymerase (e.g., T3, T7, SP6). The use and productionof an expression construct are known in the art (see, for example, WO97/32016; U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and5,804,693). If synthesized chemically or by in vitro enzymaticsynthesis, the RNA may be purified prior to introduction into the cell.For example, RNA can be purified from a mixture by extraction with asolvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof. Alternatively, the RNA may be used with no or aminimum of purification to avoid losses due to sample processing. TheRNA may be dried for storage or dissolved in an aqueous solution. Thesolution may contain buffers or salts to promote annealing, and/orstabilization of the duplex strands.

For transcription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, andpolyadenylation) may be used to transcribe the RNA strand (or strands).Therefore, in one embodiment, the nucleotide sequences for use inproducing RNA molecules may be operably linked to one or more promotersequences functional in a microorganism, a fungus or a plant host cell.Ideally, the nucleotide sequences are placed under the control of anendogenous promoter, normally resident in the host genome. Thenucleotide sequence of the present invention, under the control of anoperably linked promoter sequence, may further be flanked by additionalsequences that advantageously affect its transcription and/or thestability of a resulting transcript. Such sequences are generallylocated upstream of the operably linked promoter and/or downstream ofthe 3′ end of the expression construct and may occur both upstream ofthe promoter and downstream of the 3′ end of the expression construct,although such an upstream sequence only is also contemplated.

As used herein, the term “disease control agent”, or “gene suppressionagent” refers to a particular RNA molecule consisting of a first RNAsegment and a second RNA segment linked by a third RNA segment. Thefirst and the second RNA segments lie within the length of the RNAmolecule and are substantially inverted repeats of each other and arelinked together by the third RNA segment. The complementarity betweenthe first and the second RNA segments results in the ability of the twosegments to hybridize in vivo and in vitro to form a double strandedmolecule, i.e., a stem, linked together at one end of each of the firstand second segments by the third segment which forms a loop, so that theentire structure forms into a stem and loop structure, or even moretightly hybridizing structures may form into a stem-loop knottedstructure. The first and the second segments correspond invariably andnot respectively to a sense and an antisense sequence with respect tothe target RNA transcribed from the target gene in the target pathogenthat is suppressed by the ingestion of the dsRNA molecule. The controlagent can also be a substantially purified (or isolated) nucleic acidmolecule and more specifically nucleic acid molecules or nucleic acidfragment molecules thereof from a genomic DNA (gDNA) or cDNA library.Alternatively, the fragments may comprise smaller oligonucleotideshaving from about 15 to about 250 nucleotide residues, and morepreferably, about 15 to about 30 nucleotide residues.

As used herein, the term “genome” as it applies to cells of aplant-parasitic nematode or a host encompasses not only chromosomal DNAfound within the nucleus, but organelle DNA found within subcellularcomponents of the cell. The DNA's of the present invention introducedinto plant cells can therefore be either chromosomally integrated ororganelle-localized. The term “genome” as it applies to bacteriaencompasses both the chromosome and plasmids within a bacterial hostcell. The DNA's of the present invention introduced into bacterial hostcells can therefore be either chromosomally integrated orplasmid-localized.

As used herein, the term “plant-parasitic nematode” refers to thosenematodes that may infect, colonize, parasitize, or cause disease onhost plant material transformed to express or coated with a doublestranded gene suppression agent. As used herein, a “nematode resistance”trait is a characteristic of a transgenic plant, transgenic animal, orother transgenic host that causes the host to be resistant to attackfrom a nematode that typically is capable of inflicting damage or lossto the host. Such resistance can arise from a natural mutation or moretypically from incorporation of recombinant DNA that confersplant-parasitic nematode resistance. To impart nematode resistance to atransgenic plant a recombinant DNA can, for example, be transcribed intoa RNA molecule that forms a dsRNA molecule within the tissues or fluidsof the recombinant plant. The dsRNA molecule is comprised in part of asegment of RNA that is identical to a corresponding RNA segment encodedfrom a DNA sequence within a plant-parasitic nematode that prefers tocause disease on the host plant. Expression of the gene within thetarget plant-parasitic nematode is suppressed by the dsRNA, and thesuppression of expression of the gene in the target plant-parasiticnematode results in the plant being resistant to the nematode. Fire etal. (U.S. Pat. No. 6,506,599) generically describes inhibition of pestinfestation, providing specifics only about several nucleotide sequencesthat were effective for inhibition of gene function in the nematodespecies Caenorhabditis elegans. Similarly, US 2003/0061626 describes theuse of dsRNA for inhibiting gene function in a variety of nematodepests. US 2003/0150017 describes using dsDNA sequences to transform hostcells to express corresponding dsRNA sequences that are substantiallyidentical to target sequences in specific pests, and particularlydescribe constructing recombinant plants expressing such dsRNA sequencesfor ingestion by various plant-parasitic nematode, facilitatingdown-regulation of a gene in the genome of the target organism andimproving the resistance of the plant to the plant-parasitic nematode.

The modulatory effect of dsRNA is applicable to a variety of genesexpressed in the plant-parasitic nematode including, for example,endogenous genes responsible for cellular metabolism or cellulartransformation, including house keeping genes, transcription factors,molting-related genes, and other genes which encode polypeptidesinvolved in cellular metabolism or normal growth and development.

As used herein, the phrase “inhibition of gene expression” or“inhibiting expression of a target gene in the cell of a plant-parasiticnematode” refers to the absence (or observable decrease) in the level ofprotein and/or mRNA product from the target gene. Specificity refers tothe ability to inhibit the target gene without manifest effects on othergenes of the cell and without any effects on any gene within the cellthat is producing the dsRNA molecule. The inhibition of gene expressionof the target gene in the plant-parasitic nematode may result in novelphenotypic traits in the nematode.

The present invention provides in part a delivery system for thedelivery of the nematode control agents by ingestion of host cells orthe contents of the cells. In accordance with another embodiment, thepresent invention involves generating a transgenic plant cell or a plantthat contains a recombinant DNA construct transcribing the stabilizeddsRNA molecules of the present invention. As used herein, “taking up”refers to the process of an agent coming in contact with cells of atarget organism, such as a nematode. This may occur, for instance, bynematode feeding, by soaking, or by injection. As used herein, thephrase “generating a transgenic plant cell or a plant” refers to themethods of employing the recombinant DNA technologies readily availablein the art (e.g., by Sambrook, et al., 1989) to construct a planttransformation vector transcribing the stabilized dsRNA molecules of thepresent invention, to transform the plant cell or the plant and togenerate the transgenic plant cell or the transgenic plant that containthe transcribed, stabilized dsRNA molecules.

It is envisioned that the compositions of the present invention can beincorporated within the seeds of a plant species either as a product ofexpression from a recombinant gene incorporated into a genome of theplant cells, or incorporated into a coating or seed treatment that isapplied to the seed before planting. The plant cell containing arecombinant gene is considered herein to be a transgenic event.

The present invention provides in part a delivery system for thedelivery of disease control agents to plant-parasitic nematodes. Thestabilized dsRNA or siRNA molecules of the present invention may bedirectly introduced into the cells of a plant-parasitic nematode.Methods for introduction may include direct mixing of RNA with hosttissue for the plant-parasitic nematode, as well as engineeredapproaches in which a species that is a host is engineered to expressthe dsRNA or siRNA. In one embodiment, the RNA may be sprayed onto aplant surface. In still another embodiment, the dsRNA or siRNA may beexpressed by microorganisms and the microorganisms may be applied onto aplant surface or introduced into a root, stem by a physical means suchas an injection. In still another embodiment, a plant may be geneticallyengineered to express the dsRNA or siRNA in an amount sufficient to killthe plant-parasitic nematodes known to infest the plant.

It is also anticipated that dsRNAs produced by chemical or enzymaticsynthesis may be formulated in a manner consistent with commonagricultural practices and used as spray-on products for controllingplant disease. The formulations may include the appropriate stickers andwetters required for efficient foliar coverage as well as UV protectantsto protect dsRNAs from UV damage. Such additives are commonly used inthe bioinsecticide industry and are well known to those skilled in theart. Such applications could be combined with other spray-on insecticideapplications, biologically based or not, to enhance plant protectionfrom plant-parasitic nematodes

The present invention also relates to recombinant DNA constructs forexpression in a microorganism. Exogenous nucleic acids from which an RNAof interest is transcribed can be introduced into a microbial host cell,such as a bacterial cell or a fungal cell, using methods known in theart.

The nucleotide sequences of the present invention may be introduced intoa wide variety of prokaryotic and eukaryotic microorganism hosts toproduce the stabilized dsRNA or siRNA molecules. The term“microorganism” includes prokaryotic and eukaryotic species such asbacteria and fungi, as well as nematodes. Fungi include yeasts andfilamentous fungi, among others. Illustrative prokaryotes, bothGram-negative and Gram-positive, include Enterobacteriaceae, such asEscherichia, Erwinia, and Serratia; Bacillaceae; Rhizobiaceae, such asRhizobium; Spirillaceae, such as photobacterium, Zymomonas;Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter;Azotobacteraceae, Actinomycetales, and Nitrobacteraceae. Amongeukaryotes are fungi, such as Phycomycetes and Ascomycetes, includingSaccharomyces and Schizosaccharomyces; and Basidiomycetes, such asRhodotorula, Aureobasidium, and the like.

D. Transgenic Plants

The present invention provides seeds and plants having one or moretransgenic event. Combinations of events are referred to as “stacked”transgenic events. These stacked transgenic events can be events thatare directed at the same target organism, or they can be directed atdifferent target pathogens or pests. In one embodiment, a seed havingthe ability to express a nucleic acid provided herein also has theability to express at least one other agent, including, but not limitedto, an RNA molecule the sequence of which is derived from the sequenceof an RNA expressed in a target pathogen such as a nematode and thatforms a double stranded RNA structure upon expressing in the seed orcells of a plant grown from the seed, wherein the ingestion of one ormore cells of the plant by the target results in the suppression ofexpression of the RNA in the cells of the target.

In certain embodiments, a seed having the ability to express a dsRNA thesequence of which is derived from a target plant-parasitic nematode alsohas a transgenic event that provides herbicide tolerance. One beneficialexample of a herbicide tolerance gene provides resistance to glyphosate,N-(phosphonomethyl) glycine, including the isopropylamine salt form ofsuch herbicide.

Benefits provided by the present invention may include, but are notlimited to: the ease of introducing dsRNA into the plant-parasiticnematode cells, the low concentration of dsRNA which can be used, thestability of dsRNA, and the effectiveness of the inhibition. The abilityto use a low concentration of a stabilized dsRNA avoids severaldisadvantages of anti-sense interference. The present invention is notlimited to in vitro use or to specific sequence compositions, to aparticular set of target genes, a particular portion of the targetgene's nucleotide sequence, or a particular transgene or to a particulardelivery method, as opposed to the some of the available techniquesknown in the art, such as antisense and co-suppression. Furthermore,genetic manipulation becomes possible in organisms that are notclassical genetic models.

In order to achieve inhibition of a target gene selectively within aplant-parasitic nematode species that it is desired to control, thetarget gene should preferably exhibit a low degree of sequence identitywith corresponding genes in a plant or a vertebrate animal. Preferablythe degree of the sequence identity is less than approximately 80%. Morepreferably the degree of the sequence identity is less thanapproximately 70%. Most preferably the degree of the sequence identityis less than approximately 60%.

In addition to direct transformation of a plant with a recombinant DNAconstruct, transgenic plants can be prepared by crossing a first planthaving a recombinant DNA construct with a second plant lacking theconstruct. For example, recombinant DNA for gene suppression can beintroduced into first plant line that is amenable to transformation toproduce a transgenic plant that can be crossed with a second plant lineto introgress the recombinant DNA for gene suppression into the secondplant line.

The present invention can be, in practice, combined with other diseasecontrol traits in a plant to achieve desired traits for enhanced controlof plant disease. Combining disease control traits that employ distinctmodes-of-action can provide protected transgenic plants with superiordurability over plants harboring a single control trait because of thereduced probability that resistance will develop in the field.

The invention also relates to commodity products containing one or moreof the sequences of the present invention, and produced from arecombinant plant or seed containing one or more of the nucleotidesequences of the present invention are specifically contemplated asembodiments of the present invention. A commodity product containing oneor more of the sequences of the present invention is intended toinclude, but not be limited to, meals, oils, crushed or whole grains orseeds of a plant, or any food product comprising any meal, oil, orcrushed or whole grain of a recombinant plant or seed containing one ormore of the sequences of the present invention. The detection of one ormore of the sequences of the present invention in one or more commodityor commodity products contemplated herein is defacto evidence that thecommodity or commodity product is composed of a transgenic plantdesigned to express one or more of the nucleotides sequences of thepresent invention for the purpose of controlling plant disease usingdsRNA mediated gene suppression methods.

E. Obtaining Nucleic acids

The present invention provides methods for obtaining a nucleic acidcomprising a nucleotide sequence for producing a dsRNA or siRNA. In oneembodiment, such a method comprises: (a) analyzing one or more targetgene(s) for their expression, function, and phenotype upondsRNA-mediated gene suppression in a nematode; (b) probing a cDNA orgDNA library with a hybridization probe comprising all or a portion of anucleotide sequence or a homolog thereof from a targeted nematode thatdisplays an altered, e.g. reduced, nematode growth or developmentphenotype in a dsRNA-mediated suppression analysis; (c) identifying aDNA clone that hybridizes with the hybridization probe; (d) isolatingthe DNA clone identified in step (b); and (e) sequencing the cDNA orgDNA fragment that comprises the clone isolated in step (d) wherein thesequenced nucleic acid molecule transcribes all or a substantial portionof the RNA nucleotide acid sequence or a homolog thereof.

In another embodiment, a method of the present invention for obtaining anucleic acid fragment comprising a nucleotide sequence for producing asubstantial portion of a dsRNA or siRNA comprises: (a) synthesizingfirst and a second oligonucleotide primers corresponding to a portion ofone of the nucleotide sequences from a targeted pathogen; and (b)amplifying a cDNA or gDNA insert present in a cloning vector using thefirst and second oligonucleotide primers of step (a) wherein theamplified nucleic acid molecule transcribes a substantial portion of thea substantial portion of a dsRNA or siRNA of the present invention.

In practicing the present invention, a target gene may be derived fromH. glycines or another nematode. It is contemplated that severalcriteria may be employed in the selection of preferred target genes. TheH. glycines gene may be one which has a C. elegans ortholog with alikelihood for a strong phenotype upon RNAi knockdown of expression,including a P0 phenotype. Such targets are often those with proteinproducts involved in core cellular processes such as DNA replication,cell cycle, transcription, RNA processing, translation, proteintrafficking, secretion, protein modification, protein stability anddegradation, energy production, intermediary metabolism, cell structure,signal transduction, channels and transporters, and endocytosis. Incertain embodiments it is advantageous to select a gene for which asmall drop in expression level results in deleterious effects for thepathogen.

As used herein, the term “derived from” refers to a specified nucleotidesequence that may be obtained from a particular specified source orspecies, albeit not necessarily directly from that specified source orspecies.

In one embodiment, a gene is selected that is essentially involved inthe growth and development, of a plant-parasitic nematode. Other targetgenes for use in the present invention may include, for example, thosethat play important roles in nematode viability, growth, development,infectivity, and establishment of feeding sites. These target genes maybe one of the house keeping genes, transcription factors and the like.Additionally, the nucleotide sequences for use in the present inventionmay also be derived from homologs, including orthologs, of plant, viral,bacterial or insect genes whose functions have been established fromliterature and the nucleotide sequences of which share substantialsimilarity with the target genes in the genome of a target nematode.According to one aspect of the present invention for nematode control,the target sequences may essentially be derived from the targetedplant-parasitic nematode. Some of the exemplary target sequences clonedfrom a nematode that encode proteins or fragments thereof which arehomologues of known proteins may be found in the Sequence Listing, forinstance SEQ ID NOs:301-1026, SEQ ID NOs:1269-1702, and SEQ IDNOs:1920-1929.

For the purpose of the present invention, the dsRNA or siRNA moleculesmay be obtained by polymerase chain (PCR™) amplification of a targetgene sequences derived from a gDNA or cDNA library or portions thereof.The DNA library may be prepared using methods known to the ordinaryskilled in the art and DNA/RNA may be extracted. Genomic DNA or cDNAlibraries generated from a target organism may be used for PCR™amplification for production of the dsRNA or siRNA.

The target genes may be then be PCR™ amplified and sequenced using themethods readily available in the art. One skilled in the art may be ableto modify the PCR™ conditions to ensure optimal PCR™ product formation.The confirmed PCR™ product may be used as a template for in vitrotranscription to generate sense and antisense RNA with the includedminimal promoters. In one embodiment, the present invention comprisesisolated and purified nucleotide sequences that may be used asplant-parasitic nematode control agents. The isolated and purifiednucleotide sequences may comprise those as set forth in the sequencelisting.

As used herein, the phrase “coding sequence”, “structural nucleotidesequence” or “structural nucleic acid molecule” refers to a nucleotidesequence that is translated into a polypeptide, usually via mRNA, whenplaced under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a translation startcodon at the 5′-terminus and a translation stop codon at the3′-terminus. A coding sequence can include, but is not limited to,genomic DNA, cDNA, EST and recombinant nucleotide sequences.

The term “recombinant DNA” or “recombinant nucleotide sequence” refersto DNA that contains a genetically engineered modification throughmanipulation via mutagenesis, restriction enzymes, and the like.

For many of the plant-parasitic nematodes that are potential targets forcontrol by the present invention, there may be limited informationregarding the sequences of most genes or the phenotype resulting frommutation of particular genes. Therefore, it is contemplated thatselection of appropriate genes for use in the present invention may beaccomplished through use of information available from study of thecorresponding genes in a model organism such in C. elegans, in which thegenes have been characterized, according to the analysis described inExamples 1-8. In some cases it will be possible to obtain the sequenceof a corresponding gene from a target nematode by searching databasessuch as GenBank using either the name of the gene or the sequence from,for example, a nematode from which the gene has been cloned. Once thesequence is obtained, PCR™ may be used to amplify an appropriatelyselected segment of the gene in the target nematode for use in thepresent invention. PCR™ primers may be designed based on the sequence asfound in another organism from which the gene has been cloned. Theprimers are designed to amplify a DNA segment of sufficient length foruse in the present invention. DNA (either genomic DNA or cDNA) isprepared from the target plant-parasitic nematode, and the PCR™ primersare used to amplify the DNA segment. Amplification conditions areselected so that amplification will occur even if the primers do notexactly match the target sequence. Alternately, the gene (or a portionthereof) may be cloned from a gDNA or cDNA library prepared from aplant-parasitic nematode species, using the known gene as a probe.Techniques for performing PCR™ and cloning from libraries are known.Further details of the process by which DNA segments from targetplant-parasitic nematodes species may be isolated based on the sequenceof genes previously cloned from other species are provided in theExamples. One of ordinary skill in the art will recognize that a varietyof techniques may be used to isolate gene segments from plant-parasiticnematodes that correspond to genes previously isolated from otherspecies.

EXAMPLES

The inventors herein have identified a means for controllingplant-parasitic nematodes by providing double stranded ribonucleic acidmolecules to plant-parasitic nematodes, and a means to select sequencesthat encode these double stranded ribonucleic acid molecules. Doublestranded ribonucleic acid molecules that function upon ingestion toinhibit a biological function in a nematode may result, for example, inone or more of the following attributes: reduction in growth of anematode, inhibition of development of a nematode, or reduction ofviability. Any one or any combination of these attributes can result inan effective inhibition of plant infection or colonization, and in thecase of a plant pathogenic nematode, inhibition of plant disease, and/orreduction in severity of disease symptoms.

Example 1 Criteria for Target Gene Selection

To rank genes by likely essentiality of function in the nematodelifecycle, information from C. elegans RNA interference (RNAi)experiments available at wormbase.org was combined with additionalexperimental information. Using the >40,000 phenotype data points inwormbase, all ˜20,000 C. elegans genes were ranked for their potency ofphenotype, level of confidence in phenotype, and likelihood of effectsat multiple stages in the lifecycle. 715 C. elegans genes with BLASTamino acid homology to H. glycines of at least e-10 (see orthologydetermination below) had scores in this phenotype scoring system of 44or better, where 1 is best. Below, phenotype statistics are provided forjust the targets eventually making the top 300 list (using allcriteria). As the phenotype ranking is dependent upon currentlyavailable information, it is not meant to be all inclusive capturingevery gene that could be a good target, but rather to provide a list ofhigh quality targets likely to be successful in H. glycines RNAi basedon information in hand.

The following analysis of phenotypes evident following RNAi treatmentwas performed prior to the recent publication of Sonnichsen et al. C.elegans genome RNAi survey (Sonnichsen et al., 2005). This group tested19,075 genes by injection of dsRNA into the gonads of C. elegans wildtype strain N2 hermaphrodites and looked for effects on that animal andits progeny. They found effects for just 1,668 genes (8.7%). Providingadditional support to the robustness of the top 300 list (FIG. 3), 293were among the genes tested by Sonnichsen et al. and 257 had phenotypes(87.7%) including 176 Emb (embryonic lethal), 34 Lva (larval arrest), 34Ste (sterile), 8 Lvl (larval lethal), 2 Stp (sterile progeny), 1 Bmd(body morphological defect), 1 Dpy (dumpy), 1 Egl (egg laying abnormal).36 had wild type (WT) reports. Wild type reports increased toward thebottom of the list: 7 WT reports in 1-100, 13 WT in 101-200, 16 WT in201-300.

RNAi phenotype reports were categorized into four groups. First, reportsof lethality in the larval stages (lethal=let, larval lethal=lvl, larvalarrest=lva) were placed together and given greatest weight. Suchphenotypes if mimicked in H. glycines would be advantageous as dsRNAdelivery from the plant to the nematode would begin as the second stagejuvenile worm, a stage analogous to the dauer larva, enters the plant.Delivery would continue through development of J2, J3, and J4 juvenileor larval stages at the feeding site. Disrupting nematode physiologyduring these larval stages could prevent feeding site formation in theplant roots in the P0 (primary) generation.

Second, reports of molting (mlt), blistering (bli), and rupture (nip)defects were placed together and given second greatest weight. Suchphenotypes are rare relative to other more commonly observed effectssuch as lethality or sterility. Nematodes, including both C. elegans andH. glycines are covered by a cuticle of collagen. Molting defects couldblock shedding and reformation of the cuticle between larval stages(J2→J3, J3→J4, J4→A) and therefore developmental progression. Blisterphenotypes often also involve defects in the cuticle. Rupture phenotypesin C. elegans are not well understood but may involve loss of bodyintegrity or osmotic shifts. Rupture phenotypes are particularlyattractive for SCN control as they are likely irreversible with noopportunity for the worm to recover as may be the case for temporaryblocks to metabolism or development.

Third, sterile (ste) and embryonic (emb) lethality phenotypes wereplaced together and given third greatest weight. In C. elegans, the germline tissue, where many genes involved in fertility and embryonicdevelopment are active (maternal factors), is particularly sensitive toRNAi. In the context of an SCN infection, a sterile or embryonic lethalblock to the lifecycle alone could prevent formation of a secondgeneration but would allow initial feeding site formation and plant rootsystem damage. Fourth, other major defects in the physiology includinggrowth defective (gro), sick (sck), uncoordinated (unc), paralyzed (prl,prz) phenotypes were placed together and given fourth greatest weight.In the female H. glycines, once a feeding site is formed the wormdiscontinues moving and body wall musculature plays a limited role insurvival. However, male worms become mobile as adults so that they canseek, find, and fertilize females. Therefore, blocking of muscle andnervous system structures important for normal movement could interferewith fertilization and formation of a second generation. For thepurposes of this ranking, other non-lethal RNAi phenotypes (dumpy,short, long, body morphology defect, etc.) were not included. Phenotypesobserved by genetic mutation were also not used in this ranking system.

To rank genes based on wormbase RNAi information, genes with multiplephenotypic reports and multiple different phenotypes were favored overgenes with single phenotypic reports and single phenotypes. Multiplereports of phenotypes are helpful as they add confidence to theassignment of phenotype to a gene. Based on experiments from multiplegroups, C. elegans RNAi experiments have a false negative and falsepositive rate of ˜10-15%. Evidence of phenotypes from more than onereport decreases the likelihood of inclusion of false positives in thelist. Because C. elegans RNAi does not result in complete transcriptelimination (knockdown is estimated at 90%) and is often non-uniformthrough the population, single gene RNAi experiments often result in theobservation of multiple phenotypes over the course of the life cycle.Therefore, reporting of the phenotype of a given gene as “ste, emb, lvl”may mean that it is required throughout the lifecycle whereas aphenotype of “emb” alone may mean that the gene is only required for onestep in embryonic development. For the purposes of parasite control,genes required for multiple steps and multiple processes in thelifecycle are more attractive targets than those required at just onestage as this provides more opportunities for a dsRNA to interfere withthe lifecycle and block infection.

In each of these four categories, C. elegans RNAi reports were totaledand weighted. The first report of a phenotype in an N2 (wild type)background for a gene from a given laboratory was given a weight of 1.The second, third, etc. report of a phenotype from that same laboratorywas given a discounted weight of 0.7. Therefore, independent reportsfrom two laboratories (score=2) would be favored over two reports fromthe same laboratory (score=1.7) that might be vulnerable to a systematicerror (e.g. wrong clone selected and used twice). Reports of phenotypesin the rrf-3(−/−) genetic background which is more sensitive to RNAithan N2 were given a discounted score of 0.6.

With these categories and weightings, targets were ranked on a scalefrom a score of 1 to 44 or higher based on their tally in the matrixshown in FIG. 1. For instance, to receive a score of 3, targets had tohave a let lvl lva score of ≥2, a mlt bli nip score of X≥1, a ste embscore of ≥2, and an other phenotype score of ≥1. Most genes in C.elegans have a score worse than 44. 715 genes had a score of 44 orbetter and a potential homolog in H. glycines. For the top 300 targets,following ranking on all criteria, the average phenotype score was 22±9.The top score was a 3 (6 targets) and only 10 targets had a score lessthan 10. The most common categories were scores of 10 (61 targets with alet lvl lva score of ≥2, a mlt bli nip score of 0, a ste emb score of 2and an other phenotype score of ≥1), 15 (23 targets), 24 (26 targets),26 (93 targets), and 33 (19 targets). Averages and standard deviationsfor tallies in each category were let lvl lva score=1.9±1, mlt bli nipscore=0.3±0.7, ste emb score=2.8±1.7, and an other phenotypescore=1.6±1.3. mlt bli nip were the most rare phenotypes with only 56targets having a score>0. Totals of targets with scores ≥0 in the othercategories were 284 for let lvl lva, 281 for ste emb score of ≥2, and258 for other phenotype.

In addition to reports of visible phenotypes from wormbase, reports ofwild type (i.e. no phenotype) for each target were also recorded. Wildtype findings were viewed as a negative for target ranking and mosttargets with high numbers of wild type reports were removed fromconsideration for the top 300 list. All wild type reports were given ascore of 1 except those from Vidal et al. which were given a score of0.3 since this group's methodology seems to have resulted in a higherpercentage of wild type reports than all other reporting groups. Inaddition to total wild type tally, % wild type relative to all otherreports was also calculated. For the top 300 targets, the averages andstandard deviations for wild type tally and wild type percent were0.14±0.41 and 1.9±5.1%. 250 of the 300 targets had no wild type reports.For the 50 targets with wild type reports, the wild type tally andpercentages were 0.85±0.65 and 11.3±7.3%. In only 24 cases were wildtype reports >10% of all reports with the highest being 33%.

Next, RNAi data was compared to that available in wormbase. RNAiexperiments have been performed on more than 1,500 C. elegans genes ofinterest in feeding assays with N2, rrf-3, and other strains. Of the 715C. elegans genes under consideration with BLAST amino acid homology tothe H. glycines and wormbase phenotype scores of 44 or better (FIG. 1),information was available for about 75. Additionally, 3 genes with P0(first generation) effects in assays not on the list of 715 were addedto the list. 10 target genes of the original 715 showed no phenotype inan assay (multiple replicates with sequence confirmation of constructs)and were therefore excluded from the top 300. Phenotypes were observedfor 39 target genes in the top 300 including lvl, lva, nip, ste, emb,unc, sck and additional phenotypes not included in the ranking systemsuch as growth defective (gro), sterile progeny (stp), dumpy (dpy), andbody morphology defect (bmd).

Example 2 C. elegans P-Zero (P0) RNAi Screens

In addition to the standard RNAi assay, additional C. elegans RNAiscreens were performed. One of these was a P0 lethal screen.

P-zero (P0) or Rapid Onset RNAi Effects in C. elegans

To control Heterodera glycines by providing dsRNA from a transgenic soyplant, finding genes that are sensitive to rapid onset RNAi effectscould be useful. Such genes might block mature feeding site formation bythe J2 while slow onset genes might not show an effect until the nextgeneration. C. elegans genes where effects are seen in the exposedanimal, so called P0 (P-zero) generation effects, might predict H.glycines orthologs with high and rapid sensitivity to RNAi.

All large scale screens for RNAi effects in C. elegans have exposedinitial worms as L4s or adults (the genetic P0 (P-zero) generation) todsRNA and looked for phenotypes in their progeny, the F1 and F2generation. Sterility of the initial P0 animal was also observed in manycases. None of the microinjection screens (Gonczy et al., 2000; Piano etal. 2000; Piano et al., 2002) or feeding screens (Fraser et al., 2000;Kamath et al., 2003) note effects on the P0 animals other thansterility. Likewise, in a screen of over 1,200 C. elegans genes byfeeding from L4 start, P0 phenotypes other than sterility were notobserved. Wormbase (www.wormbase.org) records of RNAi phenotypes do noteven note the generation in which an effect is observed (P0, F1, F2,etc.) so finding P0 candidates from these records is not possible. Maedaet al. 2000 performed RNAi on 2,500 C. elegans genes by soaking of L4s.Interestingly, in the supplementary online material accompanying thepublication, Maeda et al. note 26 cases where P0 effects other thansterility were observed ranging from unhealthy to slow growing tolethality. These targets were investigated experimentally to see if anyof the genes were actually legitimate P0 effects (FIG. 2).

Of the 26 Maeda et al. entries 21 corresponded to wormbase.org entrieswhile 5 were clones of unclear origin. 19 genes have been successfullycloned into RNAi E. coli feeding constructs and sequence verified while2 had cloning difficulties. 17 of the 19 completed clones have so farbeen tested for P0 phenotypes. Experiments are in progress to finishtesting of two more clones and repeat test six others that have onlybeen tested once to confirm results. Additionally, 7 targets of interestwere tested in the same assay for a total of 24. Data was collected fromtwo life-cycle starting points. First, a P0 L4 start was performed tomimic both the Maeda assay and the standard E. coli feeding assay.Second, a P0 egg start was performed to expose the P0 animal to dsRNAthroughout development, a similar situation to that which may beencountered by a Heterodera glycines nematode upon infection of a plantproducing a dsRNA. For the egg start, the assumption is that the firstdsRNA exposure occurs as the L1 larva hatches and begins to feed.

From the 17 tested Maeda et al. (2001) candidates and 7 additionaltargets, 6 were found to have P0 phenotypes other than sterility from anegg start in the feeding assay (3 from Maeda and 3 from the additionallist). These were Y57G11C.15 (sec61 alpha), C47E12.5 (uba-1), andR07E4.6 (kin-2) from Maeda and C34G6.6 (pan-1), F52B11.3 (pan-2), andT25C8.2 (act-5) from the additional list. For the L4 start, only threegenes showed a P0 phenotypes other than sterility. The genes with L4start phenotypes were a subset of the 6 showing an egg start phenotype:Y57G11C.15 (sec61 alpha) and R07E4.6 (kin-2) from Maeda and T25C8.2(act-5) from the additional list. Of the remaining genes tested to datefrom the Maeda candidates, 4 had less severe phenotypes such assterility and F1 larval arrest while 9 are fully wild type and werelikely false positives in the Maeda screen. It remains possible howeverthat genes lacking a phenotype in a bacterial feeding assay would have aphenotype in a soaking assay.

In addition to studies with N2 C. elegans worms (laboratory standardstrain), studies were also performed for 22 of the genes in a fog-2(q71)mutant background. fog-2(q71) is a mutant that feminizes the germ lineof the hermaphrodite and is maintained as a male/female strain. Femaleworms were picked away from males as larva prior to fertilization. Suchvirgin female worms are easy to maintain for many days as they age sincethere is no F1 generation present to overgrow the plate and maytherefore aid in seeing late onset effects in the P0 adult worm. P0results observed in fog-2(q71) were identical to those seen in N2 and noadditional phenotypes were detected. fog-2 tests on subsequent geneswere therefore discontinued.

The studies showed that in C. elegans obtaining a P0 phenotype otherthan sterility from an L4 start is a very rare event but that examplesdo exist such as Y57G11C.15 (sec61 alpha), R07E4.6 (kin-2), and T25C8.2(act-5) (FIG. 2). Evidence suggests that most of the P0 phenotypes otherthan sterility recorded by Maeda et al. in their supplementary materialswere false positives. This is supported by other evidence: in additionto the present data at least six genes had been tested by otherlaboratories and found to have only wild type phenotypes in allgenerations (P0, F1, etc.). P0 phenotypes other than sterility from anL4 start are presumably rare as the adult already has many of theproteins necessary for survival and the slow kinetics of RNAi takes timeto degrade all transcripts. Genes showing an effect under suchconditions may be ones where the worm is especially sensitive to theirdisruption and therefore ranked highly among targets for consideration.P0 phenotypes other than sterility from an egg start are less rare andare seen in several of the targets of interest known to have severephenotypes (e.g. pan-1, pan-2). Presumably, a larger survey of targetswith strong phenotypes would find more.

Of the six targets in which a P0 lethal phenotype was confirmed, allhave reasonably strong orthologs among the H. glycines sequences kin-2was already confirmed as having a P0 phenotype and was therefore rankedin the top 10. pan-1 and pan-2 are both already among the top 100 usingour wormbase RNAi phenotype ranking system (FIG. 1) and additional H.glycines sequence information and will also be considered. Pan-1, whichwas missing from the ESTs, is newly identified in H. glycines based onGenome Survey sequencing. act-5, uba-1, and sec61 alpha were not yetamong the top several hundred targets using the wormbase RNAi phenotyperanking system because they lacked let, lvl, lva, mlt, bli, and nipphenotype reports in wormbase. Final rank positions of these six targetsamong the top 300 are Y57G11C.15 (sec61 alpha)=#2, C47E12.5 (uba-1)=#3,and R07E4.6 (kin-2)=#4, C34G6.6 (pan-1)=#7, F52B11.3 (pan-2)=#30, andT25C8.2 (act-5)=#21.

Example 3 C. elegans Intestine-Specific RNAi Screen

A second additional screen was also performed to aid in the selection oftarget genes (Table 1). A strain of C. elegans that is sensitive to RNAionly in the intestine was previously generated. This strain can be usedto rapidly screen the set of genes previously shown to be essential toviability and development by RNAi to identify those that are essentialspecifically in the intestine. Such genes may be advantageous targetswith plant-delivered H. glycines RNAi as the intestine is believed to bethe first tissue to come in contact with the entering dsRNA. Further,genes encoding secreted and transmembrane proteins may be vulnerable todisruption by the expression in the plant of nematocidal proteins thatdisrupt the function of these gene products.

The C. elegans intestine-specific RNAi strain was generated byintroducing a transgene that drives expression of the wild-type sid-1gene under the control of an intestine specific-promoter in an otherwisesid-1 minus background (Strain HC75). sid-1 is essential for systemicRNAi in C. elegans and encodes a transmembrane protein that is aputative dsRNA transporter (Winston, et al., 2002). Three lines, eachdriving expression from a different intestinal promoter, have beentested for sensitivity to RNAi. The promoters used are 5′ regions ofelt-2, ges-1, and ile-1. Feeding dsRNA from unc-54 (body wall muscle),dpy-7 (hypodermis), and act-1 (multiple tissues) showed no phenotype inthese strains although all three dsRNAs produced the expected phenotypein wild-type (N2) worm controls. On the other hand, feeding dsRNA fromact-5, an intestine-specific actin, and ile-1, a gene with intestinallocalization, in these strains resulted in sterility. These observationssupport the conclusion that these strains are sensitive to RNAi in theintestine but not in a number of other tissues. The ges-1::sid-1 strainwas selected for screening. Phenotypes in this strain are weaker than inN2 even for genes that only have roles in the intestine, probablybecause ges-1 expression is non-uniform or weaker than endogenous SID-1intestinal expression such that SID-1 is not always present at thelevels it would be in N2. However, phenotypes, such as 50% sterility areeasily scored relative to controls and are reproducible. 130 genes havebeen examined to date in the intestinal-RNAi strain, with a focus onsecreted and transmembrane targets and 57 have been observed to havephenotypes (Table 1). Of the 715 gene targets under consideration forthe top 300 list, 24 had phenotypes in the intestinal RNAi strainwhereas 3 were wild type. In ranking otherwise equally weighted targetspreference was given to genes showing intestinal RNAi strain phenotypesand 14 such genes made the top 300 list. The 10 that did not either hadweak intestinal RNAi phenotypes (e.g. 25% sterile) or other drawbacks(weak homology, non-orthology, etc.).

TABLE 1 C. elegans Intestinal RNAi Strain Phenotypes Intestinal RNAiIntestinal RNAi Gene Phenotype Gene Phenotype act-5 80% sterile C01G8.540% sterile Ile-1 80% sterile R13H4.4 30% sterile C16A3.3 20-70% sterileC25A11.4 40% sterile C47E12.5 40-99% sterile T26E3.3 15% sterile C48E7.620-40% sterile F54G8.3 20% sterile D1014.3 30-80% sterile ZK1058.250-80% sterile D1069.3 15-50% sterile K07D8.1 25% sterile T10H9.3 20-40%sterile H19M22.2 45% sterile T24H7.1 15-35% sterile F42C5.10 65% sterileZK973.6 20-30% sterile T03E6.7 EMB C01F1.2 15-40% sterile R03E1.2 75%sterile C54G4.8 50-80% sterile Y55H10A.1 LVA, 80% sterile F10D7.5 15-40%sterile C23H3.4 80% sterile F11G11.5 15-50% sterile F43D9.3 55% sterileF32B5.8 20-25% sterile Y57G11C.15 80% sterile F33A8.1 60-97% sterileF49C12.13 55% sterile F34D10.2 20-30% sterile T01H3.1 60% sterileF39B2.11 20-25% sterile T04A8.9 20-80% sterile F54C9.2 15-25% sterileH19M22.2 45-55% sterile F55A11.2 10-15% sterile K02B12.3 20-50% sterileF55A12.7 50-60% sterile W04A4.5 25-50% sterile F55A12.8 20% sterileY47G6A.23 25-50% sterile F55F10.1 20-25% sterile C16D9.2 30-35% sterileK07A12.3 15-25% sterile F52C6.3 35% sterile K07B1.5 25-30% sterileF53B8.1 65% sterile K07D8.1 25% sterile C49C3.11 35% sterile K12D12.240-70% sterile F41G3.4 35% sterile R04F11.2 40-70% sterile Y57G11C.3125% sterile T01B11.3 10-30% sterile

Example 4 Information on the C. elegans Gene Orthologs—PutativeMolecular Function

Besides phenotype, additional information extracted from wormbaseincluded gene name, wormpep protein ID, expression pattern, subcellularlocalization, prominent motifs, brief identification, concisedescription, and gene ontology terms. Preference for ranking was givento target genes with known molecular function over those with completelyunknown function. We observed that genes with strong phenotypes were ingeneral those with protein products involved in core cellular processessuch as DNA replication, cell cycle, transcription, RNA processing,translation including ribosome and tRNA function, protein trafficking,secretion, protein modification, protein stability and degradation,energy production including mitochondrial function, intermediarymetabolism, cell structure, signal transduction, channels andtransporters, and endocytosis. Of the 300 selected targets, 275 belongto one of these categories (Table 2). Of the top 100, 98 belonged to oneof these categories. The most frequently observed categories amongtargets with strong phenotypes were translation/ribosome/tRNAs,transcription/RNA processing, and Proteinstability/degradation/proteosome.

TABLE 2 Categorization of C. elegans 300 Top Targets by PutativeCellular Function DNA Protein replication, trafficking/ repair,secretion/ Protein modify, Transcription/ Translation/ nuclearstability/ Energy cell cycle, RNA ribosome/ import/ Protein degradation/production/ chromatin processing tRNAs export modification proteosomemitochondria totals 1-300 22 41 65 22 3 31 14 % 7% 14% 22% 7% 1% 10% 5% 1-100 7 13 14 4 2 13 8 101-200 5 14 34 6 0 11 3 201-300 10 14 17 12 1 73 Cell Receptors/ Metabolism/ structure/ Signal Other extracellulartransuction/ Enzymes matrix kinases Channels/Transporters EndocytosisOther/Unknown totals 1-300 28 21 20 1 8 25 % 9% 7% 7% 0% 3% 8%  1-100 138 11 0 6 2 101-200 8 6 4 0 1 8 201-300 7 7 5 1 1 15

Tracking of putative cellular function insures that suitable diversityis maintained in the top targets list and that the target list does notbecome too dependent upon one or a few processes which might functiondifferently in H. glycines than C. elegans. In conception, low priorityis given to developmental processes believed to be rapidly evolving innematodes such as sex determination since such processes are less likelyto be conserved between H. glycines and C. elegans. In practice, fewsuch genes scores well enough in RNAi phenotype to come intoconsideration for the top 300 list. Interestingly, considering strengthof C. elegans phenotype alone (score from 1-44 and wild type reports)even prior to inclusion of information on sequence conservation, highlyranked targets greatly over represent core cellular processes involvinglarge multi-subunit complexes such as the ribosome and proteosome. Thismay result simply from the importance of translation and proteindegradation to cellular survival. One additional interpretation of thisfinding is that subunits of large complexes tend to have strongphenotype following RNAi knockdown as they are more dosage sensitivethan proteins not acting in complexes. For example, while a monomerenzyme may retain adequate flux through a metabolic pathway with only10% of its normal protein dose, imbalance between subunit doses in alarge complex may result in complex misassembly or other functionalfailure. Recent findings in S. cerevisiae support this interpretation(Papp et al., 2003).

Example 5 Sequence Homology, Identity, and Orthology Assignment

To identify Heterodera glycines orthologs of characterized C. elegansgenes, homology searches were performed using the BLAST suite ofprograms (Altschul et al., 1990). Using predicted protein sequences forC. elegans genes (Wormpep) TBLASTN was used to search both H. glycinesclustered ESTs and other sequences available from Genbank as well asrecently generated proprietary genome survey sequences. Bitscore,e-value, and % identity were tracked. For consideration as a target, aC. elegans gene had to have a TBLASTN match to an ortholog or homolog inH. glycines with an e-value of at least e-10 or better. 715 C. elegansgenes with RNAi phenotypes ranking from 1-44 (FIG. 1) met this minimalcriterion. For these 715 genes, H. glycines matches were:

BLAST Mean ± SD Range % ID 56 ± 18  96-22 Bitscore 184 ± 141 1031-52E-value e−48 (median) 0-e−11

To build the top 300 list (FIG. 3), favorable amino acid level sequencesimilar between C. elegans and H. glycines was the second most importantfactor in ranking after RNAi phenotype. Targets features of % ID,bitscore, and e-value were divided into quartiles. Top ranking targetsin addition to having strong RNAi phenotypes also had favorable homologyfeatures indicative of orthology: bitscore (>100), e-value (<e-20), andpercentage identity (>40%). To insure orthology assignment, targets werealso tested for reciprocal BLAST matching so that when the selected H.glycines sequence was checked by BLAST back to the C. elegans wormpeplist (BLASTX), the original starting sequence was identified. 25 genesthat would otherwise have made the top 300 failed this reciprocal BLASTtest indicating that the genes in C. elegans and H. glycines wereunlikely to be orthologs. Of the final 300, 296 appeared to be clearorthologs with reciprocal BLAST top matches in both directions, 1 wastied for top match (2 close homologs in C. elegans), and 3 were within5% of the top bitscore (these were kept because of strong phenotypesamong all the top C. elegans homologs). For the top 300 genes, H.glycines matches were:

BLAST Mean ± SD Range % ID 65 ± 15  96-30 Bitscore 253 ± 158 1031-71E-value e−61 (median) 0-e−13

Of the top 300 targets, 63 appear to have been identified specificallydue to Genome Survey sequencing. Matches in the ESTs or other publiclyavailable sequences are either missing or of substantially weaker scores(mostly paralogs). The rank of these genes among the top 300 is asfollows:

23 in top 100: 1, 5, 7, 9, 15, 33, 35, 37, 38, 40, 44, 51, 53, 58, 60,67, 68, 75, 80, 82, 83, 88, 96 15 in 2nd 100: 102, 115, 134, 142, 148,151, 152, 156, 163, 164, 191, 193, 199, 200 26 in 3rd 100: 206, 208,211, 219, 226, 231, 237, 241, 242, 247, 251, 254, 256, 258, 260, 261,263, 267, 271, 272, 280, 281, 289, 292, 298, 300.

Example 6 Gene Expression in H. glycines and Other Tylenchids

The expression of target genes in H. glycines and other Tylenchid plantparasitic nematodes was also monitored. Even within H. glycines, thisprocess was imprecise since the identified transcript from the top 300list may be the actual gene of interest in the cases where the top hitwas an EST (237 out of 300) or a related homolog where the top match wasto the genomic sequence (63 out of 300). Likewise, matches in relatedTylenchids may be orthologs or homologs. Nevertheless, this informationprovides a starting point for looking at stage of expression. Expressionin J2, J3, and J4 is particularly attractive as double stranded RNAdelivery should be possible from the plant at these stages through thefeeding site while the cyst is forming. Of genes with EST representationin H. glycines, 47% were represented by a single EST. 53% of genes wererepresented by two or more ESTs. 64% had representation in stages J2,J3, or J4. 165 of the 300 targets had orthologs or homologs among ESTsand other sequencing available from other Tylenchid plant parasiticnematodes including Heterodera schachtii, Globodera rostochiensis,Globodera pallida, Meloidogyne incognita, Meloidogyne javanica,Meloidogyne arenaria, Meloidogyne hapla, Meloidogyne chitwoodi,Meloidogyne paranaensis, Pratylenchus vulnus, Pratylenchus penetrans,Radopholus similis (Table 3).

TABLE 3 Evidence of Expression of Top 300 Targets in H. glycines andother Tylenchid species. Total ESTs total H.g ESTs J2, J3, J4 H.g ESTsOther Species mean 4.7 2.3 14.0 sd 22.0 5.3 64.3 median 1 1 1 zeros 22122 135 ones 131 66 38 twos 43 46 12 three or more 102 64 113

Example 7 Target List for H. glycines RNAi

Disruption of target gene function within the plant pathogen soybeancyst nematode, Heterodera glycines, can be accomplished by RNAinterference and can result in disruption of the pathogen's lifecycle.Optimal target genes for disruption include life-cycle essential geneswhere disruption results in high penetrance death of the parasitepopulations or “genetic death” by blocking of reproduction with minimaldamage to the plant and minimal viable escaping worms reaching the nextgeneration. The inventors have provided a process for selecting RNAitargets and a list of 300 target genes predicted to be essential to H.glycines reproduction. Key features used to predict targets includeorthology to C. elegans known genes with strong and reproducible RNAinterference phenotypes. The complete top 300 target list is provided inFIG. 3. Table 4 lists selected gene targets for assaying in planttissues.

TABLE 4 Selected Gene Targets for Soybean Hairy Root and in plantaAssays Matching Nucleotide Location on Gene Name Sequence NucleotideSequence Peptide Sequence Bli-3 SeqID_1304  12-552 SeqID_1889 Bli-3SeqID_1487  12-552 SeqID_1889 C23H3.4 SeqID_510 925-735 SeqID_1056C23H3.4 SeqID_814 586-776 SeqID_1056 C26E6.4 SeqID_539 2836-3029SeqID_1051 C34G6.6 SeqID_511 3917-3429 SeqID_1233 C34G6.6 SeqID_790244-732 SeqID_1233 Cct-4 SeqID_1318 193-568 SeqID_1741 Cct-4 SeqID_1537193-568 SeqID_1741 Cgh-1 SeqID_1289 319-795 SeqID_1722 Cgh-1 SeqID_1513280-756 SeqID_1722 Cgh-1 SeqID_1289 1587-1761 SeqID_1722 Crs-1 SeqID_5351783-1487 SeqID_1035 Crs-1 SeqID_792 1353-1649 SeqID_1035 Cyc-1SeqID_513 1375-1154 SeqID_1046 Cyc-1 SeqID_804 529-750 SeqID_1046F01G10.1 SeqID_509 3122-3454 SeqID_1055 F01G10.1 SeqID_813  690-1022SeqID_1055 F52B11.3 SeqID_508 3280-3088 SeqID_1053 F52B11.3 SeqID_8111036-1228 SeqID_1053 F54B3.3 SeqID_524 2457-2222 SeqID_1058 F54B3.3SeqID_816 266-501 SeqID_1058 Kin-2 SeqID_506 1310-1023 SeqID_1032 Kin-2SeqID_787  79-366 SeqID_1032 Kin-2 SeqID_1921  15-464 SeqID_1931 Kin-2SeqID_1921 465-714 SeqID_1931 Kin-2 SeqID_1926 211-460 SeqID_1931 Kin-2SeqID_787 265-514 SeqID_1032 Kin-2 SeqID_787 514-663 SeqID_1032 Kin-2SeqID_1921 340-586 SeqID_1931 Kin-2 SeqID_1926  86-332 SeqID_1931 Kin-2SeqID_787 140-386 SeqID_1032 Let-767 SeqID_1306 328-687 SeqID_1735Let-767 SeqID_1528 295-654 SeqID_1735 Pas-4 SeqID_1292 564-894SeqID_1724 Pas-6 SeqID_1290 314-859 SeqID_1723 Ran-1 SeqID_1307 550-777SeqID_1736 Rpt-1 SeqID_1310 328-581 SeqID_1737 Rpt-1 SeqID_1531 328-581SeqID_1737 Rpt-1 SeqID_523 1356-1609 SeqID_1047 Rpt-1 SeqID_805 668-921SeqID_1047 Sec61 alpha SeqID_1330  93-488 SeqID_1751 Sec61 alphaSeqID_1548  93-488 SeqID_1751 Top-1 SeqID_1298  1-449 SeqID_1729 Top-1SeqID_1521  1-449 SeqID_1729 Top-1 SeqID_1920 1569-2017 SeqID_1930 Top-1SeqID_1925 1565-2013 SeqID_1930 Uba-1 SeqID_1923 1977-2184 SeqID_1933Uba-1 SeqID_1928 1715-1922 SeqID_1933 Uba-1 SeqID_505 2500-2708SeqID_1031 Uba-1 SeqID_786 1562-1770 SeqID_1031 Vab-10 SeqID_5422871-3759 SeqID_1028 Vab-10 SeqID_788  444-1332 SeqID_1028 Vha-12SeqID_1303  44-496 SeqID_1733 Vha-12 SeqID_1526  1-453 SeqID_1733 Vha-12SeqID_793  1-453 SeqID_1036 Vha-13 SeqID_1312  16-255 SeqID_1906 Vha-13SeqID_1533  16-255 SeqID_1906 Vha-13 SeqID_514 1979-2218 SeqID_1048Vha-13 SeqID_806 707-946 SeqID_1048 Vha-15 SeqID_519 1318-1137SeqID_1033 Vha-15 SeqID_789  997-1178 SeqID_1033 Y48B6A.3 SeqID_13021755-2384 SeqID_1732 Y48B6A.3 SeqID_1525 1697-2326 SeqID_1732 Y55H10A.1SeqID_1327 524-830 SeqID_1748 Y55H10A.1 SeqID_1545 524-830 SeqID_1748ZK1127.5 SeqID_538 285-535 SeqID_1050 ZK1127.5 SeqID_808  50-300SeqID_1050

Example 8 Selected H. glycines Genes Showing Efficacy in BlockingSoybean Cyst Nematode Infection of Soybean

Results from soaking assays show that orthologs (and fragments of suchorthologs) of pas-4 (e.g. found at nucleotides 1-544 of SEQ ID NO:1292),pas-5 (e.g. found in SEQ ID NOs:525, 569, 797, nucleotides 1-672 of SEQID NO:1293, or in SEQ ID NO:1516), pas-6 (e.g. found at nucleotides314-859 of SEQ ID NO:1290), and cgh-1 (e.g. found at nucleotides931-1567 of SEQ ID NO:1289), among others, demonstrate efficacy inblocking soybean cyst nematode (SCN) reproduction. Each of these genesare found among the top 16 entries in FIG. 3.

Selected genes or gene fragments were also tested in a soybean hairyroot assay (e.g. Narayanan et al., 1999), and plant tissues demonstratedresistance to SCN as shown in Table 5:

TABLE 5 Hairy root assay results demonstrating SCN resistance activity.Class Total Gene Name Locus Class I Class II Class III IV Exp Sec61alpha Y57G11C.15 2 1 2 0 7 Uba-1 C47E12.5 8 2 2 0 16 Kin-2 R07E4.6 20 83 4 32 Vab-10 ZK1151.1 1 1 2 0 7 Vha-15 T14F9.1 4 2 1 0 8 C34G6.6C34G6.6 1 2 0 1 7 Y48B6A.3 Y48B6A.3 0 1 0 0 2 Crs-1 Y23H5A.7 1 0 0 0 2Vha-12 F20B6.2 0 1 0 0 4 Pas-4 C36B1.4 2 0 0 0 6 Bli-3 F56C11.1 3 0 0 03 Cgh-1 C07H6.5 1 0 0 1 9 Let-767 C56G2.6 3 0 1 0 4 Pas-6 CD4.6 2 5 0 17 Ran-1 K01G5.4 2 2 0 0 5 Cyc-1 C54G4.8 1 0 0 0 2 Rpt-1 C52E4.4 2 0 0 06 Vha-13 Y49A3A.2 3 2 1 0 8 Top-1 M01E5.5 18 8 2 4 26 ZK1127.5 ZK1127.50 2 0 0 2 C26E6.4 C26E6.4 1 1 1 0 3 F52B11.3 F52B11.3 2 1 1 0 3 F01G10.1F01G10.1 6 1 3 1 10 C23H3.4 C23H3.4 2 1 0 0 5 Cct-4 K01C8.10 3 1 0 0 4F54B3.3 F54B3.3 0 1 0 0 2 Y55H10A.1 Y55H10A.1 3 1 1 0 6

Classification of Efficacy for Table 5

-   -   Class IV: greater than or equal to 50% reduction in mean cyst        number relative to mean cyst number for transgenic empty vector        wild type control;    -   Class III: 35-49% reduction in mean cyst number relative to mean        cyst number for transgenic empty vector wild type control;    -   Class II: 20-34% reduction in mean cyst number relative to mean        cyst number for transgenic empty vector wild type control    -   Class I: having individual transformation events with greater        efficacy than highest event efficacy seen with transgenic empty        vector wild type control

Transgenic soybean plants were prepared expressing fragments of genesencoding, for instance, Pas-4, Pas-5, Cgh-1, or Pas-6, and these plantswere tested for SCN resistance. Results are shown in Table 6,demonstrating efficacy in reducing SCN infection and/or reproduction.

TABLE 6 Transgenic plant analysis for SCN resistance Total Gene EventsName Locus Promoter Soy line Class I Class II Class III Class IV testedPas-4 C36B1.4 FMV Lee 74 6 3 1 0 12 A3244 6 2 0 0 Pas-5 F25H2.9 FMV Lee74 4 5 5 1 18 A3244 0 1 0 0 Pas-5 F25H2.9 E35S Lee 74 2 4 4 2 16 A3244 11 1 0 Cgh-1 C07H6.5 FMV Lee 74 4 2 2 2 11 A3244 0 2 1 1 Cgh-1 C07H6.5E35S Lee 74 5 13 2 2 27 A3244 6 2 0 2 Pas-6 CD4.6 FMV Lee 74 5 1 2 1 14A3244 1 1 2 0 Pas-6 CD4.6 E35S Lee 74 7 5 0 0 20 A3244 3 1 0 0

Classification of Efficacy for Table 6:

-   -   Class I: 10-20% reduction in mean cyst number relative to mean        cyst number for Lee 74 or A3244;    -   Class II: 20-35% reduction in mean cyst number relative to mean        cyst number for Lee 74 or A3244;    -   Class III: 36-50% reduction in mean cyst number relative to mean        cyst number for Lee 74 or A3244;    -   Class IV: >50% reduction in mean cyst number relative to mean        cyst number for Lee 74 or A3244

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

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What is claimed is:
 1. A method for controlling a plant-parasiticnematode population comprising providing an agent comprising a doublestranded ribonucleotide sequence that functions upon being taken up bythe nematode to inhibit a biological function of Pas-6 within saidnematode, wherein the double stranded ribonucleotide sequence comprisesat least 21 contiguous nucleotides of SEQ ID NO:1514 or SEQ ID NO:1290,and the complement of said at least 21 contiguous nucleotides, whereinsaid nematode comprises a Heterodera species.
 2. A method forcontrolling a plant-parasitic nematode population comprising providingan agent comprising a double stranded ribonucleotide sequence thatfunctions upon being taken up by the pathogen to inhibit a biologicalfunction of Pas-6 within said nematode, wherein said ribonucleotidesequence comprises a first ribonucleotide sequence with from about 95 toabout 100% nucleotide sequence identity along at least 21 contiguousnucleotides of a coding sequence derived from said nematode and ishybridized to a second ribonucleotide sequence that is the complement ofsaid first ribonucleotide sequence, and wherein said coding sequencederived from said nematode is SEQ ID NO:1514 or SEQ ID NO:1290, whereinsaid nematode comprises a Heterodera species.
 3. The method of claim 2,wherein said nematode comprises Heterodera glycines.
 4. A method ofcontrolling plant nematode pest infestation in a plant comprisingproviding in the diet of a plant nematode pest a dsRNA comprising: a) asense nucleotide sequence comprising at least 21 contiguous nucleotidesof a nucleic acid sequence of SEQ ID NO:1514 or SEQ ID NO:1290; and b)the complement of said sense nucleotide sequence, wherein uptake of thedsRNA by the plant nematode pest inhibits a biological function of Pas-6within said plant nematode pest, wherein said plant nematode pestcomprises a Heterodera species.
 5. The method of claim 4, wherein saiddiet comprises a plant cell transformed to express said dsRNA.
 6. Themethod of claim 1, wherein the double stranded ribonucleotide sequencecomprises at least 21 contiguous nucleotides of SEQ ID NO:1514.
 7. Themethod of claim 1, wherein the double stranded ribonucleotide sequencecomprises at least 21 contiguous nucleotides of SEQ ID NO:1290.
 8. Themethod of claim 2, wherein the first polynucleotide sequence comprisesat least 21 contiguous nucleotides of SEQ ID NO:1514.
 9. The method ofclaim 2, wherein the first polynucleotide sequence comprises at least 21contiguous nucleotides of SEQ ID NO:1290.
 10. The method of claim 4,wherein the sense nucleotide sequence comprises at least 21 contiguousnucleotides of SEQ ID NO:1514.
 11. The method of claim 4, wherein thesense nucleotide sequence comprises at least 21 contiguous nucleotidesof SEQ ID NO:1290.